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• Turowski TW, Tollervey D
• Cotranscriptional events in eukaryotic ribosome synthesis.
• Wiley Interdiscip Rev RNA. 2015; 6: 129-39
• Display abstract

• Eukaryotic ribosomes are synthesized in a complex, multistep pathway. This begins with transcription of the rDNA genes by a specialized RNA polymerase, accompanied by the cotranscriptional binding of large numbers of ribosome synthesis factors, small nucleolar RNAs and ribosomal proteins. Cleavage of the nascent transcript releases the early pre-40S and pre-60S particles, which acquire export competence in the nucleoplasm prior to translocation through the nuclear pore complexes and final maturation to functional ribosomal subunits in the cytoplasm. This review will focus on the many and complex interactions occurring during pre-rRNA synthesis, particularly in budding yeast in which the pathway is best understood.

• Simm S et al.
• Identification and Expression Analysis of Ribosome Biogenesis Factor Co-orthologs in Solanum lycopersicum.
• Bioinform Biol Insights. 2015; 9: 1-17
• Display abstract

• Ribosome biogenesis involves a large inventory of proteinaceous and RNA cofactors. More than 250 ribosome biogenesis factors (RBFs) have been described in yeast. These factors are involved in multiple aspects like rRNA processing, folding, and modification as well as in ribosomal protein (RP) assembly. Considering the importance of RBFs for particular developmental processes, we examined the complexity of RBF and RP (co-)orthologs by bioinformatic assignment in 14 different plant species and expression profiling in the model crop Solanum lycopersicum. Assigning (co-)orthologs to each RBF revealed that at least 25% of all predicted RBFs are encoded by more than one gene. At first we realized that the occurrence of multiple RBF co-orthologs is not globally correlated to the existence of multiple RP co-orthologs. The transcript abundance of genes coding for predicted RBFs and RPs in leaves and anthers of S. lycopersicum was determined by next generation sequencing (NGS). In combination with existing expression profiles, we can conclude that co-orthologs of RBFs by large account for a preferential function in different tissue or at distinct developmental stages. This notion is supported by the differential expression of selected RBFs during male gametophyte development. In addition, co-regulated clusters of RBF and RP coding genes have been observed. The relevance of these results is discussed.

• Garcia-Gomez JJ, Lebaron S, Henry Y, de la Cruz J
• Dynamics of the Spb4 interactome monitored by affinity purification.
• Methods Mol Biol. 2015; 1259: 49-67
• Display abstract

• RNA helicases constitute the largest class of NTPases involved in ribosome biogenesis, a fundamental process that has been best characterized in the eukaryotic model organism Saccharomyces cerevisiae. In yeast, genetic and biochemical analyses indicate that these RNA helicases are energy-consuming modulators of local structures inside pre-ribosomal particles that actively promote the establishment or dissociation of snoRNA:pre-rRNA base pairings, the activity of certain pre-rRNA nucleases, and/or the acquisition of pre-rRNA folds required for the recruitment or release of ribosome assembly factors and the stable assembly of ribosomal proteins. Despite significant recent advances, the precise molecular functions of RNA helicases involved in ribosome biogenesis remain largely elusive. In recent years, the purification and compositional analysis of distinct pre-ribosomal particles via affinity purification methods has been established as one of the most useful techniques to explore the yeast ribosome biogenesis pathway. In this chapter, we describe the use of different affinity purification methods to study the physical environment of RNA helicases involved in ribosome biogenesis, using as an example the putative RNA helicase Spb4 required for 60S ribosomal subunit biogenesis.

• Sharma S, Yang J, Duttmann S, Watzinger P, Kotter P, Entian KD
• Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae.
• Nucleic Acids Res. 2014; 42: 3246-60
• Display abstract

• RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m3U2634 and m3U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold-like methyltransferases and the point mutations in the S-adenosyl-L-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m5U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.

• Bassler J et al.
• A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation.
• J Cell Biol. 2014; 207: 481-98
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• Eukaryotic ribosome biogenesis involves approximately 200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a "distribution box," transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.

• Schutz S et al.
• A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.
• Elife. 2014; 3: 3473-3473
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• Within a single generation time a growing yeast cell imports approximately 14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles.

• Li YL, Li CS, Li JH
• [The research progress of RNA binding motif protein 3 biological function].
• Sheng Li Ke Xue Jin Zhan. 2014; 45: 429-33

• Rocchi L, Barbosa AJ, Onofrillo C, Del Rio A, Montanaro L
• Inhibition of human dyskerin as a new approach to target ribosome biogenesis.
• PLoS One. 2014; 9: 101971-101971
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• The product of the DKC1 gene, dyskerin, is required for both ribosome biogenesis and telomerase complex stabilization. Targeting these cellular processes has been explored for the development of drugs to selectively or preferentially kill cancer cells. Presently, intense research is conducted involving the identification of new biological targets whose modulation may simultaneously interfere with multiple cellular functions that are known to be hyper-activated by neoplastic transformations. Here, we report, for the first time, the computational identification of small molecules able to inhibit dyskerin catalytic activity. Different in silico techniques were applied to select compounds and analyze the binding modes and the interaction patterns of ligands in the human dyskerin catalytic site. We also describe a newly developed and optimized fast real-time PCR assay that was used to detect dyskerin pseudouridylation activity in vitro. The identification of new dyskerin inhibitors constitutes the first proof of principle that the pseudouridylation activity can be modulated by means of small molecule agents. Therefore, the presented results, obtained through the usage of computational tools and experimental validation, indicate an alternative therapeutic strategy to target ribosome biogenesis pathway.

• Castle CD, Sardana R, Dandekar V, Borgianini V, Johnson AW, Denicourt C
• Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae.
• Nucleic Acids Res. 2013; 41: 1135-50
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• Ribosome biogenesis is a multi-step process that couples cell growth with cell proliferation. Although several large-scale analysis of pre-ribosomal particles have identified numerous trans-acting factors involved in this process, many proteins involved in pre-rRNA processing and ribosomal subunit maturation have yet to be identified. Las1 was originally identified in Saccharomyces cerevisiae as a protein involved in cell morphogenesis. We previously demonstrated that the human homolog, Las1L, is required for efficient ITS2 rRNA processing and synthesis of the 60S ribosomal subunit. Here, we report that the functions of Las1 in ribosome biogenesis are also conserved in S. cerevisiae. Depletion of Las1 led to the accumulation of both the 27S and 7S rRNA intermediates and impaired the synthesis of the 60S subunit. We show that Las1 co-precipitates mainly with the 27S rRNA and associates with an Nsa1 and Rix1-containing pre-60S particle. We further identify Grc3 as a major Las1-interacting protein. We demonstrate that the kinase activity of Grc3 is required for efficient pre-rRNA processing and that depletion of Grc3 leads to rRNA processing defects similar to the ones observed in Las1-depleted cells. We propose that Las1 and Grc3 function together in a conserved mechanism to modulate rRNA processing and eukaryotic ribosome biogenesis.

• Das P et al.
• Insights into the mechanism of ribosomal incorporation of mammalian L13a protein during ribosome biogenesis.
• Mol Cell Biol. 2013; 33: 2829-42
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• In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.

• Gamalinda M, Jakovljevic J, Babiano R, Talkish J, de la Cruz J, Woolford JL Jr
• Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing.
• Nucleic Acids Res. 2013; 41: 1965-83
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• Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

• Ohmayer U et al.
• Studies on the assembly characteristics of large subunit ribosomal proteins in S. cerevisae.
• PLoS One. 2013; 8: 68412-68412
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• During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.

• Lu J, Sun M, Ye K
• Structural and functional analysis of Utp23, a yeast ribosome synthesis factor with degenerate PIN domain.
• RNA. 2013; 19: 1815-24
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• During synthesis of yeast ribosome, a large complex, called the 90S pre-ribosome or the small subunit processome, is assembled on the nascent precursor rRNA and mediates early processing of 18S rRNA. The Utp23 protein and snR30 H/ACA snoRNA are two conserved components of 90S pre-ribosomes. Utp23 contains a degenerate PIN nuclease domain followed by a long C-terminal tail and associates specifically with snR30. Here, we report the crystal structure of the Utp23 PIN domain at 2.5-A resolution. The structure reveals a conserved core fold of PIN domain with degenerate active site residues, a unique CCHC Zn-finger motif, and two terminal extension elements. Functional sites of Utp23 have been examined with conservation analysis, mutagenesis, and in vivo and in vitro assays. Mutations in each of three cysteine ligands of zinc, although not the histidine ligand, were lethal or strongly inhibitory to yeast growth, indicating that the Zn-finger motif is required for Utp23 structure or function. The N-terminal helix extension harbors many highly conserved basic residues that mostly are critical for growth and in vitro RNA-binding activity of Utp23. Deletion of the C-terminal tail, which contains a short functionally important sequence motif, disrupted the interaction of Utp23 with snR30 and perturbed the pre-ribosomal association of Utp23. Our data establish a structural framework for dissecting Utp23 function in the assembly and dynamics of 90S pre-ribosomes.

• Babiano R et al.
• Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles.
• Nucleic Acids Res. 2013; 41: 9461-70
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• Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

• Hierlmeier T et al.
• Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae.
• Nucleic Acids Res. 2013; 41: 1191-210
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• Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.

• Ray D et al.
• A compendium of RNA-binding motifs for decoding gene regulation.
• Nature. 2013; 499: 172-7
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• RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

• Rodriguez-Galan O, Garcia-Gomez JJ, de la Cruz J
• Yeast and human RNA helicases involved in ribosome biogenesis: current status and perspectives.
• Biochim Biophys Acta. 2013; 1829: 775-90
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• Ribosome biogenesis is a fundamental process that is conserved in eukaryotes. Although spectacular progress has been made in understanding mammalian ribosome synthesis in recent years, by far, this process has still been best characterised in the yeast Saccharomyces cerevisiae. In yeast, besides the rRNAs, the ribosomal proteins and the 75 small nucleolar RNAs, more than 250 non-ribosomal proteins, generally referred to as trans-acting factors, are involved in ribosome biogenesis. These factors include nucleases, RNA modifying enzymes, ATPases, GTPases, kinases and RNA helicases. Altogether, they likely confer speed, accuracy and directionality to the ribosome synthesis process, however, the precise functions for most of them are still largely unknown. This review summarises our current knowledge on eukaryotic RNA helicases involved in ribosome biogenesis, particularly focusing on the most recent advances with respect to the molecular roles of these enzymes and their co-factors in yeast and human cells. This article is part of a Special Issue entitled: The Biology of RNA helicases-Modulation for life.

• Uchiumi T, Baba K, Onozuka M
• [The dynamic functional structure of the ribosome discovered by the analysis of the autoimmune target].
• Seikagaku. 2013; 85: 924-31

• Filipovska A, Rackham O
• Pentatricopeptide repeats: modular blocks for building RNA-binding proteins.
• RNA Biol. 2013; 10: 1426-32
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• Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism across the eukaryotic domain. Recent computational and structural studies have provided new insights into how they recognize RNA, and show that the recognition is sequence-specific and modular. The modular code for RNA-binding by PPR proteins holds great promise for the engineering of new tools to target RNA and identifying RNAs bound by natural PPR proteins.

• Baumas K, Soudet J, Caizergues-Ferrer M, Faubladier M, Henry Y, Mougin A
• Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles.
• RNA Biol. 2012; 9: 162-74
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• Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.

• Horigome C, Mizuta K
• Ribosome biogenesis factors working with a nuclear envelope SUN domain protein: new players in the solar system.
• Nucleus. 2012; 3: 22-8
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• The nucleolus, the most prominent structure observed in the nucleus, is often called a "ribosome factory." Cells spend an enormous fraction of their resources to achieve the mass-production of ribosomes required by rapid growth. On the other hand, ribosome biogenesis is also tightly controlled, and must be coordinated with other cellular processes. Ribosomal proteins and ribosome biogenesis factors are attractive candidates for this link. Recent results suggest that some of them have functions beyond ribosome biogenesis. Here we review recent progress on ribosome biogenesis factors, Ebp2 and Rrs1, in yeast Saccharomyces cerevisiae. In this organism, Ebp2 and Rrs1 are found in the nucleolus and at the nuclear periphery. At the nuclear envelope, these proteins interact with a membrane-spanning SUN domain protein, Mps3, and play roles in telomere clustering and silencing along with the silent information regulator Sir4. We propose that a protein complex consisting Ebp2, Rrs1 and Mps3 is involved in a wide range of activities at the nuclear envelope.

• Kallberg Y, Segerstolpe A, Lackmann F, Persson B, Wieslander L
• Evolutionary conservation of the ribosomal biogenesis factor Rbm19/Mrd1: implications for function.
• PLoS One. 2012; 7: 43786-43786
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• Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-beta4-strand in RBD6 is essential for function.

• Greber BJ, Boehringer D, Montellese C, Ban N
• Cryo-EM structures of Arx1 and maturation factors Rei1 and Jjj1 bound to the 60S ribosomal subunit.
• Nat Struct Mol Biol. 2012; 19: 1228-33
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• Eukaryotic ribosome biogenesis requires many protein factors that facilitate the assembly, nuclear export and final maturation of 40S and 60S particles. We have biochemically characterized ribosomal complexes of the yeast 60S-biogenesis factor Arx1 and late-maturation factors Rei1 and Jjj1 and determined their cryo-EM structures. Arx1 was visualized bound to the 60S subunit together with Rei1, at 8.1-A resolution, to reveal the molecular details of Arx1 binding whereby Arx1 arrests the eukaryotic-specific rRNA expansion segment 27 near the polypeptide tunnel exit. Rei1 and Jjj1, which have been implicated in Arx1 recycling, bind in the vicinity of Arx1 and form a network of interactions. We suggest that, in addition to the role of Arx1 during pre-60S nuclear export, the binding of Arx1 conformationally locks the pre-60S subunit and inhibits the premature association of nascent chain-processing factors to the polypeptide tunnel exit.

• He M, Edwards BM, Kastelic D, Taussig MJ
• Eukaryotic ribosome display with in situ DNA recovery.
• Methods Mol Biol. 2012; 805: 75-85
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• Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

• Jakovljevic J et al.
• Ribosomal proteins L7 and L8 function in concert with six A(3) assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits.
• RNA. 2012; 18: 1805-22
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• Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA(3) to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA(3) pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A(3) assembly factors and for proper assembly of the neighborhoods containing domains I and II.

• Phipps KR, Charette J, Baserga SJ
• The small subunit processome in ribosome biogenesis-progress and prospects.
• Wiley Interdiscip Rev RNA. 2011; 2: 1-21
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• The small subunit (SSU) processome is a 2.2-MDa ribonucleoprotein complex involved in the processing, assembly, and maturation of the SSU of eukaryotic ribosomes. The identities of many of the factors involved in SSU biogenesis have been elucidated over the past 40 years. However, as our understanding increases, so do the number of questions about the nature of this complicated process. Cataloging the components is the first step toward understanding the molecular workings of a system. This review will focus on how identifying components of ribosome biogenesis has led to the knowledge of how these factors, protein and RNA alike, associate with one another into subcomplexes, with a concentration on the small ribosomal subunit. We will also explore how this knowledge of subcomplex assembly has informed our understanding of the workings of the ribosome synthesis system as a whole.

• Santos MC, Goldfeder MB, Zanchin NI, Oliveira CC
• The essential nucleolar yeast protein Nop8p controls the exosome function during 60S ribosomal subunit maturation.
• PLoS One. 2011; 6: 21686-21686
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• The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxyl-terminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Deltanop8/GAL::NOP8. Interestingly, Nop8p mediates association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control the exosome function during pre-rRNA processing.

• Castle CD, Cassimere EK, Lee J, Denicourt C
• Las1L is a nucleolar protein required for cell proliferation and ribosome biogenesis.
• Mol Cell Biol. 2010; 30: 4404-14
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• Ribosome biogenesis is a highly regulated process ensuring that cell growth (increase in biomass) is coordinated with cell proliferation. The formation of eukaryotic ribosomes is a multistep process initiated by the transcription and processing of rRNA in the nucleolus. Concomitant with this, several preribosomal particles, which transiently associate with numerous nonribosomal factors before mature 60S and 40S subunits are formed and exported in the cytoplasm, are generated. Here we identify Las1L as a previously uncharacterized nucleolar protein required for ribosome biogenesis. Depletion of Las1L causes inhibition of cell proliferation characterized by a G1 arrest dependent on the tumor suppressor p53. Moreover, we demonstrate that Las1L is crucial for ribosome biogenesis and that depletion of Las1L leads to inhibition of rRNA processing and failure to synthesize the mature 28S rRNA. Taken together, our data demonstrate that Las1L is essential for cell proliferation and biogenesis of the 60S ribosomal subunit.

• Belin S et al.
• Uncoupling ribosome biogenesis regulation from RNA polymerase I activity during herpes simplex virus type 1 infection.
• RNA. 2010; 16: 131-40
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• The ribosome is the central effector of protein synthesis, and its synthesis is intimately coordinated with that of proteins. At present, the most documented way to modulate ribosome biogenesis involves control of rDNA transcription by RNA polymerase I (RNA Pol I). Here we show that after infection of human cells with herpes simplex virus type 1 (HSV-1) the rate of ribosome biogenesis is modulated independently of RNA Pol I activity by a dramatic change in the rRNA maturation pathway. This process permits control of the ribosome biogenesis rate, giving the possibility of escaping ribosomal stress and eventually allowing assembly of specialized kinds of ribosomes.

• Naganuma T, Nomura N, Yao M, Mochizuki M, Uchiumi T, Tanaka I
• Structural basis for translation factor recruitment to the eukaryotic/archaeal ribosomes.
• J Biol Chem. 2010; 285: 4747-56
• Display abstract

• The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)(2)(P1)(2)(P1)(2)) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.

• Kressler D, Hurt E, Bassler J
• Driving ribosome assembly.
• Biochim Biophys Acta. 2010; 1803: 673-83
• Display abstract

• Ribosome biogenesis is a fundamental process that provides cells with the molecular factories for cellular protein production. Accordingly, its misregulation lies at the heart of several hereditary diseases (e.g., Diamond-Blackfan anemia). The process of ribosome assembly comprises the processing and folding of the pre-rRNA and its concomitant assembly with the ribosomal proteins. Eukaryotic ribosome biogenesis relies on a large number (>200) of non-ribosomal factors, which confer directionality and accuracy to this process. Many of these non-ribosomal factors fall into different families of energy-consuming enzymes, notably including ATP-dependent RNA helicases, AAA-ATPases, GTPases, and kinases. Ribosome biogenesis is highly conserved within eukaryotic organisms; however, due to the combination of powerful genetic and biochemical methods, it is best studied in the yeast Saccharomyces cerevisiae. This review summarizes our current knowledge on eukaryotic ribosome assembly, with particular focus on the molecular role of the involved energy-consuming enzymes.

• Champion EA, Kundrat L, Regan L, Baserga SJ
• A structural model for the HAT domain of Utp6 incorporating bioinformatics and genetics.
• Protein Eng Des Sel. 2009; 22: 431-9
• Display abstract

• The half-a-tetratricopeptide (HAT) repeat motif is of interest because it is found exclusively in proteins that are involved in RNA metabolism. Little is known about structure-function relationships in this class of repeat motif. Here, we present the results of a combined bioinformatics, modeling and mutagenesis study of the HAT domain of Utp6. We have derived a new HAT consensus, delineated its structure-defining residues and, by homology modeling, have placed these residues in a structural context. By considering only HAT motifs from Utp6 proteins, we identified residues that are shared by, and unique to, only this subset of HAT motifs, suggesting a key functional role. Employing both random and directed mutagenesis of the HAT domain in yeast Utp6, we have identified residues whose mutation results in loss of function. By examining these residues in the context of the homology model, we have delineated those that act by perturbing structure and those that more likely have a direct effect on function. Importantly, the residues we predict to have a direct effect on function map together on the tertiary structure, thus defining a potential functional interaction surface.

• Buyuk Y, Kocak U
• Fire-related fatalities in Istanbul, Turkey: analysis of 320 forensic autopsy cases.
• J Forensic Leg Med. 2009; 16: 449-54
• Display abstract

• In this retrospective autopsy study fire-related deaths whose autopsies were carried out in the Council of Forensic Medicine in Istanbul, Turkey were analyzed in order to evaluate the relationship between the mode of the death and the laboratory and autopsy findings. There were 320 fire-related fatalities constituting the 2.07% of all autopsy cases of that period. Of the 320 cases 228 (71.3%) were males and 91 (28.4%) were females, average age in age-determined group was 36.6 (SD: 21.98) ranging from 8 months to 98 years. Carbon monoxide intoxication played role in death of 104 cases (32.5%) either directly or together with other factors such as burning and lack of oxygen. In 32 cases (10.0%) burning was the only cause of death and in 90 cases (28.1%) burning and related complications during treatment period caused death. In 35 cases (10.9%) traumatic factors other than those related to fire were responsible for death. There were 31 cases of homicide (9.7%) and 20 cases of suicide (6.3%). Accidents constituted the majority of the cases in our autopsy population with a frequency of 51.9%. The relationship between the presence of soot in trachea or esophagus and the mode of death and that of CO-Hb and the mode of death was statistically significant. The internal findings and laboratory data of the study population were discussed particularly on the basis of the decision of vitality in burned cases.

• Melendez-Hevia E
• From the RNA world to the DNA-protein world: clues to the origin and early evolution of life in the ribosome.
• J Biosci. 2009; 34: 825-7

• Bousquet-Antonelli C, Deragon JM
• A comprehensive analysis of the La-motif protein superfamily.
• RNA. 2009; 15: 750-64
• Display abstract

• The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits.

• Nogue S, Echarte JL, Puiguriguer J, Miro O
• [About the epidemiology of intoxications].
• Rev Clin Esp. 2009; 209: 525-525

• Houmani JL, Ruf IK
• Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.
• PLoS One. 2009; 4: 5306-5306
• Display abstract

• The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

• Fujiyama-Nakamura S et al.
• Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage.
• Mol Cell Proteomics. 2009; 8: 1552-65
• Display abstract

• Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.

• Romanova L et al.
• Critical role of nucleostemin in pre-rRNA processing.
• J Biol Chem. 2009; 284: 4968-77
• Display abstract

• Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.

• McGuire AT, Keates RA, Cook S, Mangroo D
• Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae.
• Biochem Cell Biol. 2009; 87: 431-43
• Display abstract

• Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

• Horigome C, Okada T, Matsuki K, Mizuta K
• A ribosome assembly factor Ebp2p, the yeast homolog of EBNA1-binding protein 2, is involved in the secretory response.
• Biosci Biotechnol Biochem. 2008; 72: 1080-6
• Display abstract

• We have found that Ebp2p is essential for maturation of 25S rRNA and assembly of 60S pre-ribosomal subunits in Saccharomyces cerevisiae. We obtained three temperature-sensitive ebp2 mutants by PCR. Polysome analysis revealed that the synthesis of 60S ribosomal subunits was compromised in each of the ebp2 mutants at the restrictive temperature. The ebp2 alleles affected the transcriptional repression of both rRNA and ribosomal protein genes due to a secretion block. Fluorescence microscopy showed that a secretion block led to condensation of nucleolar Ebp2p, whereas that was not the case with the ebp2 mutant. These results suggest that Ebp2p is implicated in the secretory response, including changes in nucleolar architecture.

• Wu B et al.
• The solution structure of ribosomal protein S17E from Methanobacterium thermoautotrophicum: a structural homolog of the FF domain.
• Protein Sci. 2008; 17: 583-8
• Display abstract

• The ribosomal protein S17E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. S17E is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. Mammalian S17E is a phosphoprotein component of eukaryotic ribosomes. Archaeal S17E proteins range from 59 to 79 amino acids, and are about half the length of the eukaryotic homologs which have an additional C-terminal region. Here we report the three-dimensional solution structure of S17E. S17E folds into a small three-helix bundle strikingly similar to the FF domain of human HYPA/FBP11, a novel phosphopeptide-binding fold. S17E bears a conserved positively charged surface acting as a robust scaffold for molecular recognition. The structure of M. thermoautotrophicum S17E provides a template for homology modeling of eukaryotic S17E proteins in the family.

• Henras AK et al.
• The post-transcriptional steps of eukaryotic ribosome biogenesis.
• Cell Mol Life Sci. 2008; 65: 2334-59
• Display abstract

• One of the most important tasks of any cell is to synthesize ribosomes. In eukaryotes, this process occurs sequentially in the nucleolus, the nucleoplasm and the cytoplasm. It involves the transcription and processing of pre-ribosomal RNAs, their proper folding and assembly with ribosomal proteins and the transport of the resulting pre-ribosomal particles to the cytoplasm where final maturation events occur. In addition to the protein and RNA constituents of the mature cytoplasmic ribosomes, this intricate process requires the intervention of numerous protein and small RNA trans-acting factors. These transiently interact with pre-ribosomal particles at various stages of their maturation. Most of the constituents of pre-ribosomal particles have probably now been identified and research in the field is starting to unravel the timing of their intervention and their precise mode of action. Moreover, quality control mechanisms are being discovered that monitor ribosome synthesis and degrade the RNA components of defective pre-ribosomal particles.

• Hung NJ, Lo KY, Patel SS, Helmke K, Johnson AW
• Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast.
• Mol Biol Cell. 2008; 19: 735-44
• Display abstract

• We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

• Yamada H, Horigome C, Okada T, Shirai C, Mizuta K
• Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity.
• RNA. 2007; 13: 1977-87
• Display abstract

• We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.

• Liang XH, Liu Q, Fournier MJ
• rRNA modifications in an intersubunit bridge of the ribosome strongly affect both ribosome biogenesis and activity.
• Mol Cell. 2007; 28: 965-77
• Display abstract

• The presence of nucleotide modifications in rRNA has been known for nearly 40 years; however, information about their roles is sparse. Here, we describe the consequences of depleting modifications from an intersubunit bridge (helix 69) of the ribosomal large subunit in yeast. Helix 69 interacts with both A and P site tRNAs and contains five modifications. Blocking one to two modifications has no apparent effect on cell growth, whereas loss of three to five modifications impairs growth and causes the broadest defects observed thus far for modification loss in any ribosome region. Major effects include the following: (1) reduced amino acid incorporation rates in vivo (25%-60%); (2) increased stop codon readthrough activity; (3) increased sensitivity to ribosome-based antibiotics; (4) reduced rRNA levels (20%-50%), due mainly to faster turnover; and (5) altered rRNA structure in the ribosome. Taken together, the results indicate that this subset of rRNA modifications can influence both ribosome synthesis and function and in synergistic ways.

• Jenvert RM, Schiavone LH
• The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor.
• J Mol Biol. 2007; 365: 764-72
• Display abstract

• The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

• Demoinet E, Jacquier A, Lutfalla G, Fromont-Racine M
• The Hsp40 chaperone Jjj1 is required for the nucleo-cytoplasmic recycling of preribosomal factors in Saccharomyces cerevisiae.
• RNA. 2007; 13: 1570-81
• Display abstract

• Ribosome biogenesis is a major conserved cellular pathway that requires both ribosomal proteins and many preribosomal factors. Most of the pre-60S factors are recycled into the nucleus; some of them shuttle between the nucleus and the cytoplasm while a few others, like Rei1, are strictly cytoplasmic and are mostly involved in the dissociation/recycling of the pre-60S shuttling factors. Here, we investigated the role of the Jjj1 Hsp40 chaperone in ribosome biogenesis. The absence of Jjj1 leads to a cold sensitive phenotype, a defect in the relative amount of the large ribosomal subunit with the appearance of halfmers, and to cytoplasmic accumulation of shuttling factors such as Arx1 and Alb1, which stay bound to the pre-60S particles. Jjj1 is, thus, a novel pre-60S factor involved in the last cytoplasmic steps of the large ribosomal subunit biogenesis. We report the biochemical association of Jjj1 and Rei1 to similar pre-60S complexes, their two-hybrid interactions, and their functional links. Altogether, these results indicate that Rei1 and Jjj1 share many common features. However, while the functions of Jjj1 and Rei1 partially overlap, we could distinguish specific role of the two proteins in Arx1/Alb1 and Tif6 recycling. We propose that Jjj1 is preferentially required for the release of Arx1 and Alb1 shuttling factors from the cytoplasmic pre-60S particles while Rei1 is preferentially involved in their recycling.

• Xie H et al.
• Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions.
• J Proteome Res. 2007; 6: 1882-98
• Display abstract

• Identifying relationships between function, amino acid sequence, and protein structure represents a major challenge. In this study, we propose a bioinformatics approach that identifies functional keywords in the Swiss-Prot database that correlate with intrinsic disorder. A statistical evaluation is employed to rank the significance of these correlations. Protein sequence data redundancy and the relationship between protein length and protein structure were taken into consideration to ensure the quality of the statistical inferences. Over 200,000 proteins from the Swiss-Prot database were analyzed using this approach. The predictions of intrinsic disorder were carried out using PONDR VL3E predictor of long disordered regions that achieves an accuracy of above 86%. Overall, out of the 710 Swiss-Prot functional keywords that were each associated with at least 20 proteins, 238 were found to be strongly positively correlated with predicted long intrinsically disordered regions, whereas 302 were strongly negatively correlated with such regions. The remaining 170 keywords were ambiguous without strong positive or negative correlation with the disorder predictions. These functions cover a large variety of biological activities and imply that disordered regions are characterized by a wide functional repertoire. Our results agree well with literature findings, as we were able to find at least one illustrative example of functional disorder or order shown experimentally for the vast majority of keywords showing the strongest positive or negative correlation with intrinsic disorder. This work opens a series of three papers, which enriches the current view of protein structure-function relationships, especially with regards to functionalities of intrinsically disordered proteins, and provides researchers with a novel tool that could be used to improve the understanding of the relationships between protein structure and function. The first paper of the series describes our statistical approach, outlines the major findings, and provides illustrative examples of biological processes and functions positively and negatively correlated with intrinsic disorder.

• Monie TP et al.
• Structural insights into the transcriptional and translational roles of Ebp1.
• EMBO J. 2007; 26: 3936-44
• Display abstract

• The ErbB3-binding protein 1 (Ebp1) is an important regulator of transcription, affecting eukaryotic cell growth, proliferation, differentiation and survival. Ebp1 can also affect translation and cooperates with the polypyrimidine tract-binding protein (PTB) to stimulate the activity of the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). We report here the crystal structure of murine Ebp1 (p48 isoform), providing the first glimpse of the architecture of this versatile regulator. The structure reveals a core domain that is homologous to methionine aminopeptidases, coupled to a C-terminal extension that contains important motifs for binding proteins and RNA. It sheds new light on the conformational differences between the p42 and p48 isoforms of Ebp1, the disposition of the key protein-interacting motif ((354)LKALL(358)) and the RNA-binding activity of Ebp1. We show that the primary RNA-binding site is formed by a Lys-rich motif in the C terminus and mediates the interaction with the FMDV IRES. We also demonstrate a specific functional requirement for Ebp1 in FMDV IRES-directed translation that is independent of a direct interaction with PTB.

• Fuentes JL, Datta K, Sullivan SM, Walker A, Maddock JR
• In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.
• Mol Genet Genomics. 2007; 278: 105-23
• Display abstract

• The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347-456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function.

• Buchhaupt M, Kotter P, Entian KD
• Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein.
• FEMS Yeast Res. 2007; 7: 771-81
• Display abstract

• The nucleolar Saccharomyces cerevisiae protein Nep1 was previously shown to bind to a specific site of the 18S rRNA and to be involved in assembly of Rps19p into pre-40S ribosome subunits. Here we report on the identification of tma23 and nop6 mutations as recessive suppressors of a nep1(ts) mutant allele and the nep1 deletion as well. Green fluorescent protein fusions localized Tma23p and Nop6p within the nucleolus, indicating their function in ribosome biogenesis. The high lysine content of both proteins and an RNA binding motif in the Nop6p amino acid sequence suggest RNA-binding functions for both factors. Surprisingly, in contrast to Nep1p, Tma23p and Nop6p seem to be specific for fungi as no homologues could be found in higher eukaryotes. In contrast to most other ribosome biogenesis factors, Tma23p and Nop6p are nonessential in S. cerevisiae. Interestingly, the tma23 mutants showed a considerably increased resistance against the aminoglycoside G418, probably due to a structural change in the 40S ribosomal subunit, which could be the result of incorrectly folded 18S rRNA gene, missing rRNA modifications or the lack of a ribosomal protein.

• Zemp I, Kutay U
• Nuclear export and cytoplasmic maturation of ribosomal subunits.
• FEBS Lett. 2007; 581: 2783-93
• Display abstract

• Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.

• Yao W, Roser D, Kohler A, Bradatsch B, Bassler J, Hurt E
• Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2.
• Mol Cell. 2007; 26: 51-62
• Display abstract

• The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.

• Zhang J et al.
• Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.
• Genes Dev. 2007; 21: 2580-92
• Display abstract

• More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors--e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs--remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm.

• Lebreton A, Saveanu C, Decourty L, Jacquier A, Fromont-Racine M
• Nsa2 is an unstable, conserved factor required for the maturation of 27 SB pre-rRNAs.
• J Biol Chem. 2006; 281: 27099-108
• Display abstract

• In Saccharomyces cerevisiae, a large variety of pre-ribosomal factors have been identified recently, a number of which are still of unknown function. The essential pre-ribosomal 30-kDa protein, Nsa2, was characterized as one of the most conserved proteins from yeast to human. We show here that the expression of the human orthologue TINP1 complements the repression of NSA2 in yeast. Nsa2 was co-purified in several pre-ribosomal complexes and found to be essential for the large ribosomal subunit biogenesis. Like several other factors of the pre-60 S particles, the absence of Nsa2 correlated with a decrease in the 25 S and 5.8 S ribosomal RNA levels, and with an accumulation of 27 SB pre-ribosomal RNA intermediates. We show that Nsa2 is a functional partner of the putative GTPase Nog1. In the absence of Nsa2, Nog1 was still able to associate with pre-ribosomal complexes blocked in maturation. In contrast, in the absence of Nog1, Nsa2 disappeared from pre-60 S complexes. Indeed, when ribosome biogenesis was blocked upstream of Nsa2, this short half-lived protein was largely depleted, suggesting that its cellular levels are tightly regulated.

• Byard RW, Wick R, Simpson E, Gilbert JD
• The pathological features and circumstances of death of lethal crush/traumatic asphyxia in adults--a 25-year study.
• Forensic Sci Int. 2006; 159: 200-5
• Display abstract

• A 25-year retrospective study of cases of crush/traumatic asphyxia autopsied at Forensic Science SA, Adelaide, Australia from 1980 to 2004 was undertaken. A total of 79 cases of crush asphyxia was found consisting of 63 males (80%) and 16 females (20%). The age range of the males was 19-86 years (mean=41.8 years) and of the females was 19-75 years (mean=38.6 years). In 18 cases the exact circumstances of death were unclear, leaving 61 cases in which details of the fatal episode were available. Major categories included vehicle crashes (N=37), industrial accidents (N=9), farm accidents (N=6) and entrapment beneath vehicles (N=5). Forty of the 79 victims (51%) had only very minor bruises and abrasions; 28 (35%) had evidence of chest compression with rib and sternal fractures and large areas of soft tissue bruising of the chest; 7 cases (9%) had other significant injuries or findings that had contributed to death. All of these victims had signs of crush asphyxia in the form of intense purple congestion and swelling of the face and neck, and/or petechial hemorrhages of the skin of the face and/or conjunctivae. The pattern of pathological findings of crush asphyxia was not influenced by the presence or absence of concomitant serious or lethal injuries. In 4 cases (5%) where the circumstances of the lethal episode were those of crush asphyxia there were no characteristic pathological findings. This study has shown that a high percentage of crush asphyxias may be caused by vehicle accidents. It has also demonstrated that on occasion fatal crush asphyxia may have to be a diagnosis of exclusion, made only when there are characteristic death scene findings, and no evidence of lethal natural diseases or injuries at autopsy, with negative toxicological screening.

• Bleichert F, Granneman S, Osheim YN, Beyer AL, Baserga SJ
• The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation.
• Proc Natl Acad Sci U S A. 2006; 103: 9464-9
• Display abstract

• Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A(1) and/or A(2.).

• Granneman S, Bernstein KA, Bleichert F, Baserga SJ
• Comprehensive mutational analysis of yeast DEXD/H box RNA helicases required for small ribosomal subunit synthesis.
• Mol Cell Biol. 2006; 26: 1183-94
• Display abstract

• The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

• Dlakic M
• The ribosomal subunit assembly line.
• Genome Biol. 2005; 6: 234-234
• Display abstract

• Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes.

• Polzikov M, Zatsepina O, Magoulas C
• Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast.
• Biochem Biophys Res Commun. 2005; 327: 143-9
• Display abstract

• The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells.

• Dong J, Lai R, Jennings JL, Link AJ, Hinnebusch AG
• The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis.
• Mol Cell Biol. 2005; 25: 9859-73
• Display abstract

• ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

• Huang N, Negi S, Szebeni A, Olson MO
• Protein NPM3 interacts with the multifunctional nucleolar protein B23/nucleophosmin and inhibits ribosome biogenesis.
• J Biol Chem. 2005; 280: 5496-502
• Display abstract

• Protein B23/nucleophosmin is a multifunctional protein that plays roles in ribosome biogenesis, control of centrosome duplication, and regulation of p53 expression. A yeast two-hybrid screen was performed in a search for interaction partners of B23. The complementary DNA for a highly acidic protein, nucleoplasmin 3 (NPM3), was found in multiple positive clones. Protein NPM3 and its interaction with B23 were further characterized. Endogenous B23 was able to be co-immunoprecipitated with NPM3, and this complex was resistant to ribonuclease treatment and high concentrations of salt. The N-terminal 35-90 amino acids of B23 were found to be required for their interaction. Separate co-immunoprecipitation studies of B23 and NPM3 suggested the existence of two different complexes, one containing B23 and 28 S ribosomal RNA (rRNA) and another composed of B23, NPM3, and other proteins, but no RNA. NPM3 was localized in the nucleolus, and its nucleolar localization depended on active rRNA transcription. In the cells overexpressing NPM3, there were decreased rates of pre-rRNA synthesis and processing. Overexpression of a mutant of NPM3 that did not interact with B23 did not alter pre-rRNA synthesis and processing, suggesting that the interaction of NPM3 with B23 plays a role in the ribosome biogenesis.

• Dutca LM, Culver GM
• Regulation of rRNA processing: a role for a unique GTPase.
• Mol Cell. 2005; 20: 497-9
• Display abstract

• Recent findings by Karbstein et al. (2005 [this issue of Molecular Cell]) reveal that association of the preribosomal biosynthesis factors U3 snoRNA and Rcl1p is controlled by the GTPase Bms1p, suggesting that regulatory events are involved in the formation of ribosome biogenesis complexes.

• De Marchis ML, Giorgi A, Schinina ME, Bozzoni I, Fatica A
• Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation.
• RNA. 2005; 11: 495-502
• Display abstract

• In eukaryotes ribosome biogenesis required that rRNAs primary transcripts are assembled in pre-ribosomal particles and processed. Protein factors and pre-ribosomal complexes involved in this complex pathway are not completely depicted. The essential ORF YPR143W encodes in yeast for an uncharacterized protein product, named here Rrp15p. Cellular function of Rrp15p has not so far defined even if nucleolar location was referred. With the aim to define the possible role of this orphan gene, we performed TAP-tagging of Rrp15p and investigated its molecular association with known pre-ribosomal complexes. Comparative sucrose gradient sedimentation analyses of yeast lysates expressing the TAP-tagged Rrp15p, strongly indicated that this protein is a component of the pre-60S particles. Northern hybridization, primer extension and functional proteomics on TAP-affinity isolated complexes proved that Rrp15p predominately associated with pre-rRNAs and proteins previously characterized as components of early pre-60S ribosomal particles. Finally, depletion of Rrp15p inhibited the accumulation of 27S and 7S pre-rRNAs and 5.8S and 25S mature rRNA. These results provide the first indication that Rrp15p is a novel factor involved in the early maturation steps of the 60S subunits. Moreover, the identification of the protein kinase CK2 in the Rrp15p-containing pre-ribosomal particles here reported, sustains the link between ribosome synthesis and cell cycle progression.

• Karbstein K, Jonas S, Doudna JA
• An essential GTPase promotes assembly of preribosomal RNA processing complexes.
• Mol Cell. 2005; 20: 633-43
• Display abstract

• Ribosome biogenesis in eukaryotes is a highly regulated process involving hundreds of transiently associated proteins and RNAs. Although most of these assembly factors have been genetically linked to specific step(s) in the biogenesis pathway, their biochemical functions are generally unknown. Bms1, an essential protein in yeast, is the only known GTPase required for biosynthesis of the 40S ribosomal subunit and interacts with Rcl1, an essential protein suggested to be an endonuclease. Here, we show thermodynamic coupling in the binding of Bms1 to GTP, Rcl1, and U3 small nucleolar RNA (snoRNA), an essential RNA that base pairs to pre-rRNA. Rcl1 binding to preribosomes is severely limited in yeast cells expressing a Bms1 mutant defective for Rcl1 binding. Additionally, we provide evidence that the C-terminal domain of Bms1 acts as an intramolecular GTPase-activating protein. Together, these data suggest that Bms1 functions as a GTP-regulated switch to deliver Rcl1 to preribosomes, providing molecular insight into preribosome assembly.

• Killian A et al.
• Inactivation of the RRB1-Pescadillo pathway involved in ribosome biogenesis induces chromosomal instability.
• Oncogene. 2004; 23: 8597-602
• Display abstract

• Since chromosomal instability (CIN) is a hallmark of most cancer cells, it is essential to identify genes whose alteration results into this genetic instability. Using a yeast CIN indicator strain, we show that inactivation of the YMR131c/RRB1 gene, which is involved in early ribosome assembly and whose expression is induced when the spindle checkpoint is activated, alters chromosome segregation and blocks mitosis at the metaphase/anaphase transition. We demonstrate that RRB1 interacts with YPH1 (yeast pescadillo homologue 1) and other members of the Yph1 complex, RPL3, ERB1 and ORC6, involved in ribosome biogenesis and DNA replication. Transient depletion of the human homologues GRWD, Pescadillo, Rpl3, Bop1 and Orc6L resulted in an increase of abnormal mitoses with appearance of binucleate or hyperploid cells, of cells with multipolar spindles and of aberrant metaphase plates. If deregulation of proteins involved in ribosome biogenesis, commonly observed in malignant tumors, could contribute to cancer through an aberrant protein synthesis, our study demonstrates that alteration of proteins linking ribosome biogenesis and DNA replication may directly cause CIN.

• Harris R et al.
• The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR).
• J Mol Biol. 2004; 335: 573-82
• Display abstract

• In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.

• Bernstein KA, Baserga SJ
• The small subunit processome is required for cell cycle progression at G1.
• Mol Biol Cell. 2004; 15: 5038-46
• Display abstract

• Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.

• Vanrobays E, Gelugne JP, Caizergues-Ferrer M, Lafontaine DL
• Dim2p, a KH-domain protein required for small ribosomal subunit synthesis.
• RNA. 2004; 10: 645-56
• Display abstract

• Recent proteomic analyses are revealing the dynamics of preribosome assembly. Following cleavage at processing site A(2), which generates the 20S pre-rRNA (the immediate precursor to the 18S rRNA), early RRPs (ribosomal RNA processing factors) are released in bulk from the preribosomes, and the resulting pre-40S subunits are left associated with a limited set of proteins that we refer to as the SSU RRP complex. Dim2p, a core constituent of the SSU RRP complex and conserved KH-domain containing protein, is required for pre-rRNA processing and is associated with early nucleolar and late cytoplasmic pre-rRNA species. Consistently, Dim2p shuttles between the nucle(ol)us and the cytoplasm, a trafficking that is tightly regulated by growth. The association of Dim2p with the 18S rRNA dimethyltransferase Dim1p, as well as its requirement for pre-rRNA processing at cleavage sites A(1) and A(2) and for 18S rRNA dimethylation, suggest that Dim2p may recruit Dim1p to nucleolar pre-rRNAs through its KH domain.

• Emery B, de la Cruz J, Rocak S, Deloche O, Linder P
• Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae.
• Mol Microbiol. 2004; 52: 141-58
• Display abstract

• The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre-60S ribosomal complexes. Here, we show that Has1p is an essential trans-acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature-sensitive has1-1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre-rRNA processing by pulse-chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre-rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.

• Hage AE, Tollervey D
• A surfeit of factors: why is ribosome assembly so much more complicated in eukaryotes than bacteria?
• RNA Biol. 2004; 1: 10-5
• Display abstract

• Recent years have seen a dramatic increase in the number of ribosome synthesis factors identified in the yeast Saccharomyces cerevisiae. Most of these are not predicted to directly catalyze either RNA processing or modification, and they are therefore predicted to function in some sense as assembly factors, promoting the assembly and/or disassembly of the processing and modification machinery, binding of the ribosomal proteins and correct folding of the pre-rRNAs and rRNAs. In contrast, ribosome synthesis in E. coli, which has also been extensively analyzed, appears to involve a very small number of potential assembly factors. Here we will consider the differences between eukaryotic and bacterial ribosome synthesis that may underlie this distinction.

• Karger B, Lorin de la Grandmaison G, Bajanowski T, Brinkmann B
• Analysis of 155 consecutive forensic exhumations with emphasis on undetected homicides.
• Int J Legal Med. 2004; 118: 90-4
• Display abstract

• A total of 155 consecutive forensic exhumations performed in Munster, Germany from 1967 to 2001 were evaluated retrospectively on the basis of the autopsy report, the police report and the death certificate. Histology and toxicology were performed in most cases. The postmortem intervals varied from 8 days to 8 years. Compared to other countries, the autopsy rate was low (1.2-1.4%) and the exhumation rate high (0.016%): principle of reciprocity. The cause of death could be clearly determined in 103 cases (66.5%) and histology or toxicology were decisive in 40%. Some findings were discernable using immunohistochemistry after considerable postmortem intervals, such as acute myocardial infarction after 1 year and pneumonia after 2 years and a diazepam intoxication was determined after 4.5 years. Major deviations between the cause of death as stated on the death certificate and as diagnosed after autopsy existed in 57 cases (37%). A more detailed analysis revealed five subgroups. 1. primary suspicion of intoxication (n=18) confirmed in 6 cases including 3 homicides (with parathion, clozapin, diazepam) which are described in more detail. 2. primary suspicion of homicide other than poisoning (n=51) confirmed in 19 cases. There was a serial killing of 15 patients by injection of air. In the remaining 4 cases, a shaken infant, craniocerebral injuries from blows with beer bottles, a craniocerebral gunshot and a multiplicity of blunt force injuries were diagnosed. The latter two cases are described in more detail. Superficial external examinations and the low autopsy rate were 2 common reasons for the occurrence of "buried homicides" (n=22)-not a single forensic autopsy had been performed directly after the death of the victims. 3. primary suspicion of medical malpractice (n=39). 4. accidents including traffic accidents (n=30). 5. clarification of the cause of death, circumstances or identity (n=17). Exhumations were frequently successful for recovering evidence which should better have been collected immediately after the death of an individual. Exhumations can also be regarded as an instrument to evaluate the quality of death certificates and death investigations.

• Rosado IV, de la Cruz J
• Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.
• RNA. 2004; 10: 1073-83
• Display abstract

• Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles. The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown. To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p. In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele. This screen identified the so far uncharacterized Npa1p (YKL014C). Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of halfmer polysomes in the slowly growing npa1-1 mutant. Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs. In addition, 27SA(2) pre-rRNA is prematurely cleaved at site C(2). Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles. We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits.

• Vos HR, Bax R, Faber AW, Vos JC, Raue HA
• U3 snoRNP and Rrp5p associate independently with Saccharomyces cerevisiae 35S pre-rRNA, but Rrp5p is essential for association of Rok1p.
• Nucleic Acids Res. 2004; 32: 5827-33
• Display abstract

• Biogenesis of eukaryotic ribosomal subunits proceeds via a series of precursor ribonucleoprotein particles that correspond to different stages in the maturation pathway. The different pre-ribosomal particles each contain a distinct complement of non-ribosomal, trans-acting factors that are crucial for correct and efficient progress of the maturation process. Although in recent years we have gained considerable insight into the composition of the pre-ribosomal particles, our knowledge how the ordered association with and their dissociation from the pre-ribosome of these trans-acting factors is controlled is still quite limited. Here, we have studied the mutual dependence between three of these factors, Rrp5p, U3 snoRNP and Rok1p, all essential for the early stages of pre-rRNA processing/assembly, for association with the 35S pre-rRNA in Saccharomyces cerevisiae. Using co-immunoprecipitation assays, we show that Rrp5p and U3 snoRNP associate independently of each other and that the two factors do not detectably interact prior to incorporation into the pre-ribosome. In contrast, association of the putative RNA helicase Rok1p, which is known to genetically interact with Rrp5p, is absolutely dependent on the presence of the latter protein but does not require U3.

• Anantharaman V, Aravind L
• Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability.
• BMC Genomics. 2004; 5: 45-45
• Display abstract

• BACKGROUND: The emergence of eukaryotes was characterized by the expansion and diversification of several ancient RNA-binding domains and the apparent de novo innovation of new RNA-binding domains. The identification of these RNA-binding domains may throw light on the emergence of eukaryote-specific systems of RNA metabolism. RESULTS: Using sensitive sequence profile searches, homology-based fold recognition and sequence-structure superpositions, we identified novel, divergent versions of the Sm domain in the Scd6p family of proteins. This family of Sm-related domains shares certain features of conventional Sm domains, which are required for binding RNA, in addition to possessing some unique conserved features. We also show that these proteins contain a second previously uncharacterized C-terminal domain, termed the FDF domain (after a conserved sequence motif in this domain). The FDF domain is also found in the fungal Dcp3p-like and the animal FLJ22128-like proteins, where it fused to a C-terminal domain of the YjeF-N domain family. In addition to the FDF domains, the FLJ22128-like proteins contain yet another divergent version of the Sm domain at their extreme N-terminus. We show that the YjeF-N domains represent a novel version of the Rossmann fold that has acquired a set of catalytic residues and structural features that distinguish them from the conventional dehydrogenases. CONCLUSIONS: Several lines of contextual information suggest that the Scd6p family and the Dcp3p-like proteins are conserved components of the eukaryotic RNA metabolism system. We propose that the novel domains reported here, namely the divergent versions of the Sm domain and the FDF domain may mediate specific RNA-protein and protein-protein interactions in cytoplasmic ribonucleoprotein complexes. More specifically, the protein complexes containing Sm-like domains of the Scd6p family are predicted to regulate the stability of mRNA encoding proteins involved in cell cycle progression and vesicular assembly. The Dcp3p and FLJ22128 proteins may localize to the cytoplasmic processing bodies and possibly catalyze a specific processing step in the decapping pathway. The explosive diversification of Sm domains appears to have played a role in the emergence of several uniquely eukaryotic ribonucleoprotein complexes, including those involved in decapping and mRNA stability.

• Kazmin DA, Chinenov Y, Larson E, Starkey JR
• Comparative modeling of the N-terminal domain of the 67kDa laminin-binding protein: implications for putative ribosomal function.
• Biochem Biophys Res Commun. 2003; 300: 161-6
• Display abstract

• Laminin-binding protein/p40 (LBP/p40) precursor appears to be involved in two seemingly unrelated activities-cell adhesion and ribosomal biogenesis. Analysis of primary structure revealed a two-domain organization of the LBP/p40. The N-terminal portion of LBP is similar to the S2 family of prokaryotic ribosomal proteins, while the C-terminus is unique for Metazoa and is involved in extraribosomal functions. To gain insight into putative ribosomal functions of LBP we performed comparative modeling of the N-terminal domain using crystal structures of S2p from Thermus thermophilus. The LBP model assumes an alpha-beta sandwich fold similar to that of S2. Modeling revealed the loss of a significant portion of ribosomal RNA (rRNA) interaction domain, lack of conservation of many residues involved in interactions with rRNA, and a major shift in surface charge distribution (compared to the S2 protein). The overall stability of the fold argues against a proposed transmembrane domain in the central part of the protein. Partial overlap in S2 and laminin-binding domains suggests that ribosomal and surface receptor functions would be mutually exclusive. The possible biological role of LBP/p40 bifunctionality is discussed.

• Elif D, Akgur SA, Ozturk P, Sen F
• Fatal poisonings in the Aegean region of Turkey.
• Vet Hum Toxicol. 2003; 45: 106-8
• Display abstract

• Between January 1996 and November 2000, 4,251 autopsies were performed at the Council of Forensic Medicine, Morgue Specialization Office, Izmir, Turkey. Among these medicolegal autopsies, 331 fatal poisoning cases (206 men and 125 women) were evaluated retrospectively for their legal investigation results, autopsy findings, and reports of toxicological analyses. The most common cause of fatal poisoning was insecticides (43%). Then followed carbon monoxide (27%) and alcohol (20%). Among the insecticides, the organophosphorus insecticides comprised 78%; all insecticide poisoning cases were suicidal oral ingestions. In fatal alcohol poisoning cases the cause of death was ethanol, methanol and both with 34, 15 and 51% respectively. Drug related deaths were rare.

• Milkereit P, Kuhn H, Gas N, Tschochner H
• The pre-ribosomal network.
• Nucleic Acids Res. 2003; 31: 799-804
• Display abstract

• Recent achievements in yeast functional proteomics have significantly advanced our knowledge about ribosome biogenesis. Here, we present a program developed to integrate data from various proteome analyses with cell biological data on components present in the ribosome producing factories. This program allows users to attribute factors to certain complexes and to specific steps of ribosome biogenesis. Thus, it helps to gain novel insights into the complex network involved in maturation of ribosomal subunits. The database can be accessed at the URL http://www.pre-ribosome.de.

• Pitula J, Ruyechan WT, Williams N
• Two novel RNA binding proteins from Trypanosoma brucei are associated with 5S rRNA.
• Biochem Biophys Res Commun. 2002; 290: 569-76
• Display abstract

• We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei which we have termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18-amino-acid insert in the N-terminal region of p37. These two proteins have been localized to the nucleus based on immunofluorescence microscopy. To gain insight into their function, we have utilized UV crosslinking, coimmunoprecipitation, and sucrose density gradients to identify T. brucei RNA species that associate with p34 and p37. These experiments have demonstrated a specific interaction of both p34 and p37 with the 5S ribosomal RNA and indicate that other RNA species are unlikely to be specifically bound. This suggests a role for p34 and p37 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis.

• Bjork P et al.
• A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis.
• Mol Biol Cell. 2002; 13: 3683-95
• Display abstract

• Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

• Eschrich D, Buchhaupt M, Kotter P, Entian KD
• Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis.
• Curr Genet. 2002; 40: 326-38
• Display abstract

• The essential Saccharomyces cerevisiae gene YLR186w [ NEP1 (nucleolar essential protein 1), also recently named EMG1 (essential for mitotic growth 1)] is highly conserved in eukaryotes and archaea. In S. cerevisiae, Nep1p is localized in the nucleus with a rod-shaped morphology, which first suggested a spindle/microtubule association for ScNEP1 (S. cerevisiae NEP1). However, in Candida albicans and in HeLa cells, Nep1p is a protein of the nucleoli. CaNEP1 (C. albicans NEP1) and HsNEP1 (Homo sapiens NEP1) heterologously complement the essential phenotype in a S. cerevisiae nep1 deletion mutant. However, the ScNEP1 spindle/microtubule phenotype is not found with HsNEP1 and CaNEP1, which shows that the spindle/microtubule association is specific for ScNep1p and that it is not the primary essential function of Nep1p. A temperature-sensitive ScNEP1 (ts1) allele was isolated and revealed a strongly increased sensitivity to paromomycin, a translational inhibitor which binds to RNA, indicating that ribosome biogenesis within the nucleolus is probably affected. This was confirmed by polysome profile and ribosomal subunit analysis, showing a ribosomal subunit imbalance with a decrease in 40S subunits, due to reduced amounts of native 18S rRNA. Furthermore, ScSAM2, encoding S-adenosylmethionine synthetase, was isolated as a multicopy-suppressor of the ScNEP1 (ts1) mutant allele and medium supplementation with S-adenosylmethionine restored its growth. For the first time, these results identified a novel protein with an essential function in ribosomal biogenesis which directly or indirectly interferes with a methylation reaction during the early steps of pre-rRNA processing necessary for the generation of 40S ribosomal subunits.

• Ospina JK, Matera AG
• Proteomics: the nucleolus weighs in.
• Curr Biol. 2002; 12: 2931-2931
• Display abstract

• Numerous studies have indicated that the nucleolus is involved in a variety of cellular processes besides its well-known function in ribosome biosynthesis. A recent study describing the nucleolar proteome opens the way to new avenues of research on this important nuclear suborganelle.

• Milic J, Nikolic S, Mihailovic Z
• [Analysis of causes of death in long-term survivors of injuries sustained in traffic accidents].
• Srp Arh Celok Lek. 2002; 130: 149-53
• Display abstract

• INTRODUCTION: The manner of death, i.e. if death is moros or violent, is the most important fact for the court and therefore, the most important part of the finding of autopsy reports [1]. To recognize the manner of death in cases with long outliving period after injury could be difficult for forensic pathologists. In such cases, the dissector should be able to point out the direct relationship between initial injury and death by using his (her) own experience and medical knowledge. Could the deaths provoked by low injuries be prevented? These injuries have score of 3 by Abbreviated Injury Scale (AIS) i.e. 12-20 by injury Severity Score (ISS) [3-5]. PURPOSE: The purpose of this paper is to suggest the measures for improvement of postmortem autopsy diagnosis of causes of death in cases with long outliving period (more than 15 days) after initial traffic injury. MATERIAL AND METHOD: A retrospective autopsy study was performed. It included the material of the Institute of Forensic Medicine in Belgrade for 1998. The autopsy report and accessible clinical medical data were analyzed for persons fatally injured in traffic accidents who outlived trauma more than 15 days. The sample was statistically prepared (chi 2 test, correlation coefficient). RESULTS AND DISCUSSION: The sample included 31 persons injured in traffic accidents with outliving period longer than 15 days: 21 males and 10 females (chi 2 = 0.047; p > 0.1). Average age was 49.90 years (SD = 18.28). All persons in our sample were over the age of 19. The most commonly injured persons were pedestrians (16). The mean outliving period was 41.19 days (SD = 12.60). There was a weak positive correlation between outliving period and age in our sample (coefficient of linear correlation r = 0.35). The authors combined the autopsy and available clinical data in order to get the ISS value for each case. The mean ISS value was 36.18 (SD = 8.70). There was no correlation between outliving period and severity of trauma (coefficient of linear correlation r < 0.14). All deaths in our sample were violent according to autopsy reports. In autopsy reports, dissectors always noted only one injured body region: head and neck injuries in 21 cases, chest injuries in 3, trauma of locomotor system in 5 and in 2 cases abdominal injuries. However, by analyzing these reports, the authors emphasized that in 22 cases one body region was severely injured, in 7 cases two body regions and three regions in 2 cases. According to the authors severe injury has score 3 or more by 3. In four cases the dissectors pointed no complication of initial injuries as a competitive cause of death. In 15 cases they mentioned it as general, and in the rest of cases as decided (e.g. pneumonia, sepsis, thromboembolism, etc.). In five cases, the complications of initial injury were the precipitated and immediate cause of death (the initial injury in all these cases was less than 16 by ISS i.e. severe but not critical). The seven cases were treated microscopically. These microscopical findings only proved the already established microscopical autopsy findings and were not crucial for case solution. It was alarming, that one third of cases in our sample were completed without considering the clinical medical data. This is forensic vitium artis. Nowadays, there are a few syndromes which could be the cause of death i.e. fat embolism syndrome [7], multiple organ failure) [8, 9] and systemic inflammatory response syndrome [9, 10]. The diagnosis of these syndromes is possible only clinically: the autopsy and histological findings are not specific. CONCLUSION: As long as a direct chain of events can be traced from the injury to the death, then the initial injury must be considered to be the basic cause of death, and this fact may have profound legal implications for both civil compensation and criminal responsibility. Some of the most difficult problems in forensic pathology concern deaths from which posttraumatic complications are disputed as being fatal causative factors. The agony and dying are irreversible dynamic pathophysiological processes. By autopsy only the morphological consequences of these processes could be noted by dissector. The dynamics of dying, direct correlation between initial injury and death, as well as appearance and development of complications provoked by trauma could be established only by clinical medical data. Therefore, medical clinical data are crucial for forensic pathologists and for solving the problems about the mode and manner of death in cases with long outliving period. Microscopical findings have only academic and scientific importance and are less useful in daily practice. The authors suggest that all complications of injury must be generally involved in autopsy reports, and all severe injuries should separately be registered both in medical data and autopsy reports. The finding of cause of death must include all observed severe injuries and not only one of the most severe injuries and its complications.

• Brodersen DE, Clemons WM Jr, Carter AP, Wimberly BT, Ramakrishnan V
• Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA.
• J Mol Biol. 2002; 316: 725-68
• Display abstract

• We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.

• Moghadamnia AA, Abdollahi M
• An epidemiological study of poisoning in northern Islamic Republic of Iran.
• East Mediterr Health J. 2002; 8: 88-94
• Display abstract

• We examined the causes and mortality of poisoning in the province of Mazandaran. In all, 1751 poisoning cases referred to four main hospitals over a three-year period (1997-2000) were included. More poisoning cases were females (55.5%) than males (45.5%) but the proportional mortality for males was greater than for females (65% versus 35%). The greatest proportion of poisonings occurred between the ages of 16 and 25 years. Most frequent was intentional poisoning, followed by accidental and occupational poisoning. Medicines were the most common cause, followed by chemicals such as pesticides. Poisoning by opiates, aluminium or zinc phosphide, rodenticides, petroleum and ethanol intoxication was also observed. Pesticide poisoning was most frequently fatal.

• Wehner KA, Baserga SJ
• The sigma(70)-like motif: a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis.
• Mol Cell. 2002; 9: 329-39
• Display abstract

• Little is understood about the role of nucleolar RNA binding proteins in ribosome biogenesis, although there is a clear need for them based on the strict folding requirements of the pre-rRNA. We have identified a superfamily of RNA binding proteins whose members are required for different stages of ribosome biogenesis. The Imp4 superfamily is composed of five individual families (Imp4, Rpf1, Rpf2, Brx1, and Ssf) that all possess the sigma(70)-like motif, a eukaryotic RNA binding domain with prokaryotic origins. The Imp4 superfamily members associate with RNAs that are consistent with their distinct roles in ribosome biogenesis and suggest the mechanisms by which they function.

• Chodorowski Z, Wiergowski M, Sein Anand J
• [Fatal intoxication with paramethoxyamphetamine].
• Przegl Lek. 2002; 59: 379-80
• Display abstract

• From August to December of the year 2000 we ascertained 7 rapid deaths because of acute intoxication with tablets colloquially called Ufo. There were five men and two women aged from 20 to 29 (average 24) years. Analytic tests showed that one tablet of Ufo contains 40 mg of paramethoxyamphetamine, 1.0 mg of amphetamine, 0.05 mg of methamphetamine, 0.03 mg of 3-4-methylenedioxymethamphetamine. The levels of paramethoxyamphe-tamine in the blood of the deceased, determined by gas chromatography, were from 0.68 to 10.0 (approx. 3.2) mg/L and in the urine from 16 to 64 (approx. 35) mg/L. The press and television campaign caused elimination of fatal intoxications with parametoxy-amphetamine during the last 13 months.

• Takaku H, Mutoh E, Horiuchi H, Ohta A, Takagi M
• Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa.
• Biochem Biophys Res Commun. 2001; 284: 194-202
• Display abstract

• Cycloheximide (CYH) resistance in Candida maltosa is dependent on the induction of a ribosomal protein, Q-type L41, the 56th residue of which is glutamine, not proline as in ordinary P-type L41. We found that a 38-kDa protein in a wild-type C. maltosa ribosomal fraction became undetectable upon CYH treatment but detectable again with the establishment of CYH resistance by the induction of Q-type L41. We cloned a gene coding for this protein and named it RAY38 (ribosome-associated protein of yeast). Ray38p is a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, and has a putative RNA-binding motif RGG. The ribosome-associated Ray38p was phosphorylated at serine and threonine residues, and Ray38p that was dissociated from ribosome by CYH treatment was highly phosphorylated in threonine residues. A ray38 null mutant recovered faster from CYH-caused growth stasis than the wild-type strain, suggesting that the dissociation of Ray38p from ribosome facilitates the induction of CYH resistance in C. maltosa.

• Park JH, Jensen BC, Kifer CT, Parsons M
• A novel nucleolar G-protein conserved in eukaryotes.
• J Cell Sci. 2001; 114: 173-185
• Display abstract

• We describe here a novel, evolutionarily conserved set of predicted G-proteins. The founding member of this family, TbNOG1, was identified in a two-hybrid screen as a protein that interacts with NOPP44/46, a nucleolar phosphoprotein of Trypanosoma brucei. The biological relevance of the interaction was verified by co-localization and co-immunoprecipitation. TbNOG1 localized to the trypanosome nucleolus and interacted with domains of NOPP44/46 that are found in several other nucleolar proteins. Genes encoding proteins highly related to TbNOG1 are present in yeast and metazoa, and related G domains are found in bacteria. We show that NOG1 proteins in humans and Saccharomyces cerevisae are also nucleolar. The S. cerevisae NOG1 gene is essential for cell viability, and mutations in the predicted G motifs abrogate function. Together these data suggest that NOG1 may play an important role in nucleolar functions. The GTP-binding region of TbNOG1 is similar to those of Obg and DRG proteins, which, together with NOG, form a newly recognized family of G-proteins, herein named ODN. The ODN family differs significantly from other G-protein families, and shows several diagnostic sequence characteristics. All organisms appear to possess an ODN gene, pointing to the biological significance of this family of G-proteins.

• Berlin IuA
• [Ribosome changes a root].
• Bioorg Khim. 2001; 27: 397-400

• Hong B, Wu K, Brockenbrough JS, Wu P, Aris JP
• Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae.
• Nucleic Acids Res. 2001; 29: 2927-37
• Display abstract

• Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth at 37 degrees C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide. Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37 degrees C. Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus. Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect. All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature. Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions. Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected. Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.

• Nikolic S, Micic J, Mihailovic Z
• [Correlation between survival time and severity of injuries in fatal injuries in traffic accidents].
• Srp Arh Celok Lek. 2001; 129: 291-5
• Display abstract

• INTRODUCTION: In forensic pathology, only trauma systems based on disintegration of anatomic structure of organs and tissues, could be used for objectivization, comparison and establishing of severity of injuries. Trauma systems based on pathophysiological values are useless. The Abbreviated Injury Scale (AIS) and its derivate Injury Severity Score (ISS) are the most common. AIS coded injuries are divided into six body regions and injuries are assigned a six-digit score in relation to their severity. ISS results the sum of the squares of the highest AIS values from the three most severely injured body regions. In this way, the ISS values are discontinued and vary from 0 (absence of injuries) to 75 (incompatible-with-life injury). PURPOSE: The purpose of this paper is to establish the correlation degree between outliving period and trauma severity in persons fatally injured in traffic accidents, and according to this finding to point out the ISS value of critical injury. MATERIAL AND METHOD: A retrospective autopsy study was performed; it included the material of the Institute of Forensic Medicine in Belgrade of 1998. The autopsy reports and accessible clinical medical data were analyzed for persons over the age of 18, fatally injured in traffic accidents who survived trauma less than 15 days. The sample was statistically prepared (chi 2-test, t-test, correlation coefficient, regression line). RESULTS AND DISCUSSION: The sample included 272 persons: 193 males and 79 females. The proportion of men was more significant (chi 2 = 4.76; 0.01 < p < 0.05). Average age was 51.08 years (SD = 18.08): males 49.84 +/- 17.41 and females 54.09 +/- 19.38. The most frequently injured persons in our sample were pedestrians (134). The authors combined the autopsy and accessible clinical data in order to obtain the ISS value for each case. They considered that all persons found dead on the spot or died ante portam did not outlive trauma. The sample distribution by ISS values showed three peaks: for ISS--75, 41-50 and 26-35. Peaks indicated the number of the injured body regions and trauma severity in these persons. In 87 persons who did not survive, the ISS value was 75. There were 73 persons without outliving period with ISS values less than 75: their mean ISS value was 31.87 (SD = 11.30). In 112 cases the mean outliving period was 4.79 days (SD = 3.77) and their mean ISS value was 18.05 (SD = 15.33), which was a statistically significant lower ISS value than in previous group (t = 7.015; p < 0.001). A weak negative correlation between outliving period and ISS values in our sample was noted (coefficient of linear correlation r = -0.452). Our sample is representative (t = 8.37). Coefficient of a determination (r2 = 0.20), pointed to the fact that direct correlation outliving period-trauma severity was only about 20% and the rest of correlation i.e. 80% depended on other factors (e.g. effective emergency medical system and triage, prompt and correct diagnosis, adequate medical treatment and care, etc.). The calculated linear regression was as follows: outliving period approximately 52-3 ISS. This regression pointed out that critical and potentially fatal injury, in our sample, was injury with ISS of 17. There were 22 persons with ISS < or = 7. Six of them died on the spot as car passengers; they died due to mechanical asphyxia (thoracoabdominal pressure) or respiratory and/or circulation failure due to critical chest injury (flail chest, contusions and rupture of the lungs with consequent haemopneumothorax). The rest of 16 persons survived trauma in an average of 8.56 days (SD = 3.88), and the causes of death were pneumonia, thrombus and fat embolism, sepsis, etc. CONCLUSION: By anaylzing our sample of fatally injured persons in traffic accidents (unpenetrated blunt trauma), there was a negative weak correlation between the outliving period and severity of injury based on ISS. This correlation was partly direct but mostly depended on other factors (e.g. effective emergency medical system and triage, prompt and correct diagnosis, adequate medical treatment and care, etc.). Establishment of these factors could be possible through state medical projects in big medical trauma centres. Prospect registration, evaluation and scoring of all injuries in hospitals and dissecting rooms, and comparison of the obtained results, can give valid data on mortality of injured people, bad diagnosis, and appropriate medical treatment. The autopsy of injured persons dead on the spot can point out what kind of injuries are incompatible with life, as well as with their severity. The autopsy of injured persons who survived trauma can point to the most frequent injury complications, clinical diagnosis and preventable deaths. According to this paper, the critical injury by ISS is 17. In such cases, the forensic pathologist must answer the following questions: whether the death was due to trauma; whether the precipitated cause of death was the consequence or complication of injury; what were the mechanism and mode of dying; whether the death was preventable; if there were possible malpractice and negligence, etc.

• Brunel C, Ehresmann B, Ehresmann C, McKeown M
• Selection of genomic target RNAs by iterative screening.
• Bioorg Med Chem. 2001; 9: 2533-41
• Display abstract

• A growing number of proteins are known to exert their regulatory or biological functions via RNA binding. In some cases genetic interactions allow us to infer candidate targets for RNA directed regulation, but in many other cases identification of potential regulatory targets is problematic. We have developed an in vitro biochemical screen, SETIS (SElection of <> Target RNAs by Iterative Screening) that allows screening of a major portion of the genome for identification of potential targets for RNA binding proteins.

• Leary DJ, Huang S
• Regulation of ribosome biogenesis within the nucleolus.
• FEBS Lett. 2001; 509: 145-50
• Display abstract

• Ribosome biogenesis is both necessary for cellular adaptation, growth, and proliferation as well as a major energetic and biosynthetic demand upon cells. For these reasons, ribosome biogenesis requires precise regulation to balance supply and demand. The complexity of ribosome biogenesis gives rise to many steps and opportunities where regulation could take place. For trans-acting factors involved in ribosome biogenesis in the nucleolus, there may be a dynamic coordination, both spatially and temporally, that regulates their functions from the transcription of rDNA to the assembly and export of preribosomal particles. Here we summarize most of the described regulations on ribosome biogenesis in the nucleolus. However, these may represent only a small fraction of a larger picture. Further studies are required to determine the initial signals, signal transduction pathways utilized, and the specific targets of these regulatory modifications and how these are used to control ribosome biogenesis as a whole.

• Daugeron MC, Kressler D, Linder P
• Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p.
• RNA. 2001; 7: 1317-34
• Display abstract

• Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly.

• Wade C, Shea KA, Jensen RV, McAlear MA
• EBP2 is a member of the yeast RRB regulon, a transcriptionally coregulated set of genes that are required for ribosome and rRNA biosynthesis.
• Mol Cell Biol. 2001; 21: 8638-50
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• In an effort to identify sets of yeast genes that are coregulated across various cellular transitions, gene expression data sets derived from yeast cells progressing through the cell cycle, sporulation, and diauxic shift were analyzed. A partitioning algorithm was used to divide each data set into 24 clusters of similar expression profiles, and the membership of the clusters was compared across the three experiments. A single cluster of 189 genes from the cell cycle experiment was found to share 65 genes with a cluster of 159 genes from the sporulation data set. Many of these genes were found to be clustered in the diauxic-shift experiment as well. The overlapping set was enriched for genes required for rRNA biosynthesis and included genes encoding RNA helicases, subunits of RNA polymerases I and III, and rRNA processing factors. A subset of the 65 genes was tested for expression by a quantitative-relative reverse transcriptase PCR technique, and they were found to be coregulated after release from alpha factor arrest, heat shock, and tunicamycin treatment. Promoter scanning analysis revealed that the 65 genes within this ribosome and rRNA biosynthesis (RRB) regulon were enriched for two motifs: the 13-base GCGATGAGATGAG and the 11-base TGAAAAATTTT consensus sequences. Both motifs were found to be important for promoting gene expression after release from alpha factor arrest in a test rRNA processing gene (EBP2), which suggests that these consensus sequences may function broadly in the regulation of a set of genes required for ribosome and rRNA biosynthesis.

• Wegierski T, Billy E, Nasr F, Filipowicz W
• Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast.
• RNA. 2001; 7: 1254-67
• Display abstract

• Maturation of 18S rRNA and biogenesis of the 40S ribosomes in yeast requires a large number of trans-acting factors, including the U3 small nucleolar ribonucleoprotein (U3 snoRNP), and the recently characterized cyclase-like protein Rcl1p. U3 snoRNP is a key particle orchestrating early 35S rRNA cleavage events. A unique property of Rcl1p is that it specifically associates with U3 snoRNP, but this association appears to occur only at the level of nascent ribosomes and not with the U3 monoparticle. Here we report the characterization of Bms1p, a protein that associates with Rcl1p in multiple structures, including a specific complex sedimenting at around 10S. Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein, and its depletion interferes with processing of the 35S pre-rRNA at sites A0, A1, and A2, and the formation of 40S subunits. The N-terminal domain of Bms1p has structural features found in regulatory GTPases and we demonstrate that mutations of amino acids implicated in GTP/GDP binding affect Bms1p activity in vivo. The results indicate that Bms1p may act as a molecular switch during maturation of the 40S ribosomal subunit in the nucleolus.

• Harnpicharnchai P et al.
• Composition and functional characterization of yeast 66S ribosome assembly intermediates.
• Mol Cell. 2001; 8: 505-15
• Display abstract

• The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.

• Nakamoto K et al.
• Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.
• Am J Pathol. 2001; 159: 1363-74
• Display abstract

• F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.

• Milkereit P et al.
• Maturation and intranuclear transport of pre-ribosomes requires Noc proteins.
• Cell. 2001; 105: 499-509
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• How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known. Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport. They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic. Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing. Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing. We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation.

• Gleizes PE et al.
• Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex.
• J Cell Biol. 2001; 155: 923-36
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• To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

• Becam AM, Nasr F, Racki WJ, Zagulski M, Herbert CJ
• Ria1p (Ynl163c), a protein similar to elongation factors 2, is involved in the biogenesis of the 60S subunit of the ribosome in Saccharomyces cerevisiae.
• Mol Genet Genomics. 2001; 266: 454-62
• Display abstract

• RIA1 (YNL163c) is a quasi-essential gene that encodes a protein with strong similarities to elongation factors 2. Small C-terminal deletions in the protein lead to a severe growth defect. In the case of a 22-residue C-terminal deletion this can be suppressed by intragenic mutations in the RIA1 gene or dominant extragenic mutations in TIF6, which is thought to be involved in the biogenesis of the 60S subunit of the ribosome. The dominant TIF6 alleles can also suppress the phenotype associated with a complete deletion of the RIA1 gene. Depletion of Ria1p has a dramatic effect on the polysome profile: there is a severe reduction in the level of the 80S monosomes, an imbalance in the 40S/60S ratio, and halfmers appear. Dissociation of the monosomes and polysomes in the Ria1p depletion mutant revealed a specific reduction in the amount of 60S subunits. Localization experiments with HA-tagged derivatives of Ria1p did not detect any stable association of Ria1p with ribosome subunits, 80S monosomes or polysomes. Cell fractionation experiments show that Ria1p is found in both the cytoplasmic fraction and the nuclear fraction. Taken together, these data suggest that Ria1p is involved in the biogenesis of the 60S subunit of the ribosome.

• Schreinzer D, Frey R, Stimpfl T, Vycudilik W, Berzlanovich A, Kasper S
• Different fatal toxicity of neuroleptics identified by autopsy.
• Eur Neuropsychopharmacol. 2001; 11: 117-24
• Display abstract

• Autopsies and toxicological analyses at the Institute of Forensic Medicine revealed 85 fatal intoxications with neuroleptics in Vienna from 1991 to 1997. A total of 17 cases were linked to a single neuroleptic (NL) alone, while 68 deaths were attributed to a combination of NLs with other drugs. The most frequently detected agent was prothipendyl (n=41). During the study period the number of defined daily doses of high-potency NLs prescribed increased significantly (P< or =0.001) due to increased prescribing of new atypical antipsychotics. The quantity of intermediate- and low-potency NLs dispensed remained stable. The most frequently prescribed NL was haloperidol. The relative toxicities of different NLs were calculated by dividing the number of deaths caused by this NL into the number of defined daily doses prescribed in the observation period (f-value). Single-substance intoxications and multiple-substance intoxications were distinguished. The highest f-values were associated with low-potency NLs, especially with prothipendyl, chlorprothixene and levomepromazine. Low f-values were found for the group of high-potency NLs, including flupentixol, fluphenazine, haloperidol and pimozide, as well as olanzapine. Compared to the f-values for all NLs prescribed, f-values for low-potency NLs were shown to be significantly higher concerning single-substance intoxications (P< or = 0.05) and multiple-substance intoxications (P < or = 0.001), while f-values for high-potency NLs were significantly lower (P< or = 0.05 and P< or = 0.001). We are not aware of the psychiatric diagnoses in our post-mortem sample. However, the present results indicate that careless use of low-potent NLs should be avoided in patients with a potential risk of accidental or suicidal overdose.

• Mayer C, Suck D, Poch O
• The archaeal homolog of the Imp4 protein, a eukaryotic U3 snoRNP component.
• Trends Biochem Sci. 2001; 26: 143-4
• Display abstract

• Homologs of the Imp4 protein, a component specific to the eukaryotic U3 snoRNP complex, have been found in all archaeal genomes. The archaeal and eukaryotic Imp4 proteins that are related to four other protein families, the Imp4-like, the SSF1 homologs and two sets of hypothetical proteins, are characterized by the Imp4 signature pattern. These findings, together with the presence of other snoRNPs homologs in Archaea, provide evidence for similar RNA processing and folding in Eukarya and Archaea.

• Huber MD, Dworet JH, Shire K, Frappier L, McAlear MA
• The budding yeast homolog of the human EBNA1-binding protein 2 (Ebp2p) is an essential nucleolar protein required for pre-rRNA processing.
• J Biol Chem. 2000; 275: 28764-73
• Display abstract

• The human EBP2 protein was found by two-hybrid analysis to interact with the Epstein-Barr virus nuclear antigen 1 (EBNA1). Homologs of human EBP2 can be found in Caenorhabditis elegans, Schizosaccharomyces pombe, and in Saccharomyces cerevisiae, and they all share a conserved 200-300-amino acid block of residues at their C termini. To understand the cellular function of EBP2, we have begun to study the protein in S. cerevisiae. The yeast Ebp2 protein contains N-terminal, nucleolar-associated KKE motifs, and deletion analysis reveals that the C-terminal conserved region is required for the activity of the protein. The EBP2 gene codes for an essential protein that localizes to the nucleolus. Temperature-sensitive ebp2-1 mutants become depleted of ribosomes and cease to divide after several generations at the restrictive temperature of 36 degrees C. This decline in ribosome levels is accompanied by a diminution in the levels of the 35 S-derived recombinant RNAs (rRNAs) (in particular the 25 S and 5.8 S rRNAs). Pulse-chase, Northern, and primer extension analysis of the rRNA biosynthetic pathway indicates that ebp2-1 mutants are defective in processing the 27 SA precursor into the 27 SB pre-rRNA.

• Agalarov SC, Sridhar Prasad G, Funke PM, Stout CD, Williamson JR
• Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain.
• Science. 2000; 288: 107-13
• Display abstract

• The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.

• Rodriguez-Gabriel MA, Remacha M, Ballesta JP
• The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0.
• J Biol Chem. 2000; 275: 2130-6
• Display abstract

• Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L. infantum, some of them inducing an osmosensitive phenotype at 37 degrees C. The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes. The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype. In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions. These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved.

• Tsuno A, Miyoshi K, Tsujii R, Miyakawa T, Mizuta K
• RRS1, a conserved essential gene, encodes a novel regulatory protein required for ribosome biogenesis in Saccharomyces cerevisiae.
• Mol Cell Biol. 2000; 20: 2066-74
• Display abstract

• A secretory defect causes specific and significant transcriptional repression of both ribosomal protein and rRNA genes (K. Mizuta and J. R. Warner, Mol. Cell. Biol. 14:2493-2502, 1994), suggesting the coupling of plasma membrane and ribosome syntheses. In order to elucidate the molecular mechanism of the signaling pathway, we isolated a cold-sensitive mutant with a mutation in a gene termed RRS1 (regulator of ribosome synthesis), which appeared to be defective in the signaling pathway. The rrs1-1 mutation greatly reduced transcriptional repression of both rRNA and ribosomal protein genes that is caused by a secretory defect. RRS1 is a novel, essential gene encoding a nuclear protein of 203 amino acid residues that is conserved in eukaryotes. A conditional rrs1-null mutant was constructed by placing RRS1 under the control of the GAL1 promoter. Rrs1p depletion caused defects in processing of pre-rRNA and assembly of ribosomal subunits.

• Sugita C, Sugiura M, Sugita M
• A novel nucleic acid-binding protein in the cyanobacterium Synechococcus sp. PCC6301: a soluble 33-kDa polypeptide with high sequence similarity to ribosomal protein S1.
• Mol Gen Genet. 2000; 263: 655-63
• Display abstract

• Cyanobacteria are prokaryotes that carry out plant-type photosynthesis and contain several eukaryotic-type RNA-binding proteins. Using a single-stranded DNA column, a 33-kDa protein was isolated and characterized from Synechococcus sp. PCC6301. This protein of 293 amino acids is similar in overall structure to the ribosomal protein S1 found in the same species, and contains three repeated units that are highly similar to the S1 motif originally found in the ribosomal protein S1 of Escherichia coli. However, the 33-kDa protein was found not to be associated with ribosomes and its nucleic acid binding specificity is distinct from that of the ribosomal protein S1. As this protein has high affinity for both single- and double-stranded DNA, as well as for poly(G) and poly(A), we tentatively named it nucleic acid-binding protein 1 (Nbp1).

• Poole A, Jeffares D, Penny D
• Early evolution: prokaryotes, the new kids on the block.
• Bioessays. 1999; 21: 880-9
• Display abstract

• Prokaryotes are generally assumed to be the oldest existing form of life on earth. This assumption, however, makes it difficult to understand certain aspects of the transition from earlier stages in the origin of life to more complex ones, and it does not account for many apparently ancient features in the eukaryotes. From a model of the RNA world, based on relic RNA species in modern organisms, one can infer that there was an absolute requirement for a high-accuracy RNA replicase even before proteins evolved. In addition, we argue here that the ribosome (together with the RNAs involved in its assembly) is so large that it must have had a prior function before protein synthesis. A model that connects and equates these two requirements (high-accuracy RNA replicase and prior function of the ribosome) can explain many steps in the origin of life while accounting for the observation that eukaryotes have retained more vestiges of the RNA world. The later derivation of prokaryote RNA metabolism and genome structure can be accounted for by the two complementary mechanisms of r-selection and thermoreduction.

• Lyman SK, Gerace L, Baserga SJ
• Human Nop5/Nop58 is a component common to the box C/D small nucleolar ribonucleoproteins.
• RNA. 1999; 5: 1597-604
• Display abstract

• We have identified an apparent human homolog of the yeast Nop5/Nop58 protein. hNop5/Nop58 codes for a protein of predicted molecular weight 59.6 kDa and is 46.8% identical to Saccharomyces cerevisiae Nop5/Nop58. Immunofluorescent staining with antibodies against hNop5/Nop58 indicate that it is localized primarily to the nucleolus, and coimmunoprecipitation from nuclear extracts demonstrates that hNop5/Nop58 interacts with the box C/D family of snoRNAs. Thus, hNop5/Nop58 is a common component of the box C/D snoRNPs, and joins fibrillarin as the second such component identified and characterized in metazoans.

• Eppens NA, Rensen S, Granneman S, Raue HA, Venema J
• The roles of Rrp5p in the synthesis of yeast 18S and 5.8S rRNA can be functionally and physically separated.
• RNA. 1999; 5: 779-93
• Display abstract

• The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.

• Dechampesme AM, Koroleva O, Leger-Silvestre I, Gas N, Camier S
• Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway.
• J Cell Biol. 1999; 145: 1369-80
• Display abstract

• A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

• Ho JH, Johnson AW
• NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae.
• Mol Cell Biol. 1999; 19: 2389-99
• Display abstract

• A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.

• Huang C
• [Analysis of 3130 cases of medicolegal postmortem].
• Fa Yi Xue Za Zhi. 1998; 14: 22-4
• Display abstract

• 3130 cases of medicolegal postmortem in Shantou City between January 1977 and December 1996 were analyzed statistically. The results revealed that the ratio of male to female was 2.3 and most dead were young and middle age. The number of postmortem has increased in resent years. Of 3130 cases, 2829 (92.3%) were caused by mechanical injury, mechanical asphyxia, sudden death and poisoning. As to manners of the death, 1226 were related to accidents (39.2%), 776 homicide(29.8%), 658 suicide (21.0%). These results were compared with other studies reported in domestic and international literature.

• Daugeron MC, Linder P
• Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.
• RNA. 1998; 4: 566-81
• Display abstract

• Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit.

• Jonasson B, Jonasson U, Saldeen T
• The manner of death among fatalities where dextropropoxyphene caused or contributed to death.
• Forensic Sci Int. 1998; 96: 181-7
• Display abstract

• Dextropropoxyphene (DXP) is one of the most prescribed analgesic compounds in Sweden. To investigate the manner of death among fatalities where DXP caused or contributed to death all medico-legal autopsies performed in Sweden in 1992-1996 were analysed on the bases of toxicological analyses and death certificates. DXP in peripheral blood was found in 1782 (7.5%) of the total 23,691 blood samples. According to the death certificates 956 (54%) of the 1782 cases were classified as fatal DXP poisoning. Among these, the manner of death was classified as accidental in 49 cases (5%), suicidal in 542 cases (57%) and undetermined in 365 cases (38%). The reported manner of death differed between the six forensic medicine districts in Sweden. The accident rate differed significantly between the district with the highest rate (9%) and the districts with the lowest rate (1%). One district had a significantly higher incidence of suicide (73%) than four of the other districts, while another district had a significantly lower incidence of suicide (33%) than all the other districts. The accident classification rate among the physicians performing ten or more autopsies varied from 0% to 17%, the suicide classification rate from 25% to 83% and the rate of undetermined manner of death from 8% to 71%. A major conclusion drawn from this study is that accidental DXP fatalities may be underestimated. This may have serious consequences, as under-reporting of accidental DXP fatalities will increase the risk that knowledge of the high toxicity of DXP will not reach the population consuming this drug. Since valid death statistics concerning the manner of death at DXP fatalities are needed to provide the base for preventive actions, special attention should be paid to the classification process, in order to increase the uniformity of the assessments among the different physicians, and to avoid under-reporting of accidents.

• Frisk M
• [Accidents, suicide or cry for help? 173 fatal cases on the railroads during 1990-95].
• Lakartidningen. 1997; 94: 973-4

• Szathmary E
• Origins of life. The first two billion years.
• Nature. 1997; 387: 662-3

• Brinkmann B
• [Errors in autopsy in Germany. Results of a multicenter study (II)].
• Arch Kriminol. 1997; 199: 65-74
• Display abstract

• In the result of a workshop held in Munster on 1st May 1993, 23 institutes of legal medicine in Germany reported death cases where the primary supposition was natural death or a death without legal relevance, but where the autopsies by chance revealed an unnatural death or a death with legal relevance. In the institutes participating in the study 13,000 autopsies were performed during the period of the reports, i.e. 75% of all legal autopsies performed in one year in Germany. A total of 92 "discoveries by chance" were reported where a natural death was given on the death certificate after the external examination of the corpse among them 49 accidents, 10 homicides, and 19 deaths by medical implications. In 717 deaths the manner of death was certified as "unclear" on the death certificate, but the autopsy revealed an unnatural manner of death. Some of these cases are also "discoveries by chance" after serious medical errors during the examination of corpses. Among these 717 cases were 35 homicides. It seems remarkable, that the prosecutors did not order a toxicological examination in several cases of fatal intoxications. Furthermore the results show that doctors often overlooked signs of carbon monoxide poisoning during the external examination of corpses. From errors made during the external examination another group of cases resulted which were initial classified as unnatural death without legal relevance, but the autopsy surprisingly revealed relevant facts. Finally in 29 autopsies carried out after exhumation evidence of homicide was found in 3 cases. On the basis of this multicentre study it can be estimated that there are at least 1200 homicide cases or 11,000 cases of unnatural death per year which do not appear in the official statistics and have been classified as natural death. The considerable grey area of fatal cases in connection with medical intervention resulting from the study (n > 2000) is also alarming. In addition at least 100 undiscovered homicides are certainly included in the group of death cases certified as unclear during the external examination and where no autopsy was performed. Sources of the grey area are essentially errors made during the external examination of corpses and to a lesser degree incorrect decisions to release the corpse, mistakes were seldom made also by doctors performing the autopsy. The study shows a considerable grey area of unnatural death cases, which exists within the modern society of Germany. In face of this emphasis must placed on the historical demands of criminologists and forensic pathologists: reinforced education and further education of medical students and doctors in the field of the external examination of corpses. Introduction of a specialized medical external examination of corpses Adherence to the 3 classical classifications of the manner of death: natural, unnatural and unclear on the death certificate. Increased number of autopsies.

• Jeeninga RE, Van Delft Y, de Graaff-Vincent M, Dirks-Mulder A, Venema J, Raue HA
• Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA.
• RNA. 1997; 3: 476-88
• Display abstract

• The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation.

• Wahren CA, Allebeck P, Rajs J
• Unnatural causes of death among drug addicts in Stockholm: an analysis of health care and autopsy records.
• Subst Use Misuse. 1997; 32: 2163-83
• Display abstract

• The aim of this study was to assess causes of death among 258 unnaturally deceased drug addicts who had been part of an earlier longitudinal follow-up of 1,630 patients treated in Stockholm County. An evaluation of the causes of death was carried out on the basis of hospital records and autopsy protocols. The majority of deaths were caused by accidents (39%) and suicides (31%). Intoxications were involved in 67 of the 101 accidents. Violent methods were employed in 40 of the 79 suicides. An exceptionally large proportion (28%) of the fatalities occurred during or immediately after hospitalization, which calls for increased attention to this patient group by the health services.

• Zanchin NI, Roberts P, DeSilva A, Sherman F, Goldfarb DS
• Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.
• Mol Cell Biol. 1997; 17: 5001-15
• Display abstract

• The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

• Yeh LC, Deshmukh M, Woolford JL, Lee JC
• Involvement of lysine 270 and lysine 271 of yeast 5S rRNA binding protein in RNA binding and ribosome assembly.
• Biochim Biophys Acta. 1996; 1308: 133-41
• Display abstract

• Contributions of the highly conserved K270 and its neighboring K271 in the C-terminal region of the yeast ribosomal protein L1 to 5S rRNA binding and ribosome assembly were examined by in vivo and in vitro studies on the consequences of 14 substitution mutations. All mutant proteins with a single amino-acid substitution at either position were able to bind 5S rRNA in vitro to an extent comparable to the wild-type. Yeast cells expressing these mutant proteins, except the K270G mutant, grew at nearly normal rates. Mutations of K270 appeared to produce more demonstrable effects than those of K271. The double mutant K270,271G bound RNA poorly and yeast cells expressing the mutant protein grew 30% slower. Double mutants K270,271E and K270,271R were lethal, although the mutant protein was assembled into the 60S ribosomal subunits. The resultant subunits were not stable leading eventually to cell death. The in vitro RNA binding ability of the respective protein was reduced by 60% and 20%. Taken together, the present data identified K270 and K271 as important amino-acid residues in the function of the yeast ribosomal protein L1.

• Bode W et al.
• The metzincin-superfamily of zinc-peptidases.
• Adv Exp Med Biol. 1996; 389: 1-11

• Bubunenko MG, Schmidt J, Subramanian AR
• Protein substitution in chloroplast ribosome evolution. A eukaryotic cytosolic protein has replaced its organelle homologue (L23) in spinach.
• J Mol Biol. 1994; 240: 28-41
• Display abstract

• The chloroplast translational system differs from the eubacterial ones in containing several ribosomal proteins (RPs) that have no apparent homologues in eubacteria, and in having their RP genes distributed in two cellular genome compartments. The genes maintained in the organelle genome encode mainly ribosome assembly proteins. The discovery in spinach and related plants (Caryophyllidae) of a disrupted chloroplast gene encoding the ribosome assembly protein L23 raised speculations about the transfer of the functional rpl23 gene to the nucleus or the evolutionary loss of L23 protein requirement. To solve this problem, we overexpressed in E. coli the intact rpl23 gene from corn (Zea mays), purified the protein and raised antibodies. Based on immunoanalysis, we show that a prokaryotic-type L23 protein is absent in spinach. Concomittantly we have isolated a new protein from spinach chloroplast 50 S ribosomal subunits and determined its amino acid sequence. The data revealed an unexpectedly high sequence identity to the eukaryotic family of cytosolic L23 proteins (reported from yeast, trypanosome and rat), with conservation of a peptide motif responsible for the specific interaction of these proteins with domain III of 26 S and 23 S rRNA. We propose that the prokaryotic-type L23 protein in the chloroplast ribosomes of Caryophyllidae has been replaced by a homologue of the eukaryotic cytosolic L23 family. These results represent the first case of a protein (gene) substitution in chloroplast ribosome evolution, and open a new view on how the nuclear genome could progressively exert stronger control over the chloroplast translational system. We also describe experiments on the incorporation of chloroplast L23 into E. coli ribosomes, its effect on cell growth, and an unexpected immuno cross-reaction between two chloroplast RP families.

• Tollervey D, Lehtonen H, Jansen R, Kern H, Hurt EC
• Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly.
• Cell. 1993; 72: 443-57
• Display abstract

• We have generated temperature-sensitive lethal point mutations in the small nucleolar RNA-associated protein fibrillarin (encoded by the NOP1 gene in yeast) and analyzed their effects on ribosome synthesis. The five alleles tested all prevent synthesis of normal ribosomes, but in dramatically different ways. At the non-permissive temperature, the nop1.2 and nop1.5 alleles prevent synthesis of both 18S and 25S rRNA and all pre-rRNA species except the 35S primary transcript. In contrast, the nop1.3, nop1.4, and nop1.7 alleles do not strongly impair processing. In nop1.3 strains, nucleolar methylation of pre-rRNA is strongly inhibited; late, cytoplasmic methylation of 18S rRNA and tRNA methylation continue. The nop1.4 and nop1.7 alleles result in the synthesis of cytoplasmic 60S ribosomal subunits with strongly aberrant mobilities on sucrose gradients even at the permissive temperature, owing to the impairment of a late step in ribosome assembly. Thus, all major posttranscriptional activities in ribosome synthesis, pre-rRNA processing, pre-rRNA modification, and ribosome assembly are dependent on fibrillarin.

• Lilleng P, Morild I
• [Causes of death among alcoholics].
• Tidsskr Nor Laegeforen. 1993; 113: 2556-60
• Display abstract

• During the five year period 1987-91, 377 deaths among alcoholics were examined at the Gade Institute, Department of Forensic Medicine, University of Bergen. The material was dominated by male alcoholics. More than half the cases were regarded as natural deaths. Among these, the dominating cause of death was cardiovascular disease. The rest of the material, where death was attributed to non-natural causes, was dominated by lethal intoxications. A large number of the deaths were suicides.

• Ripmaster TL, Vaughn GP, Woolford JL Jr
• A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly.
• Proc Natl Acad Sci U S A. 1992; 89: 11131-5
• Display abstract

• We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which there is a deficit of 60S ribosomal subunits. Cold sensitivity and the assembly defect are recessive and cosegregate, defining a single essential gene that we designated DRS1 (deficiency of ribosomal subunits). The wild-type DRS1 gene was cloned by complementation of the cold-sensitive phenotype of drs1. Sequence analysis reveals a high degree of similarity to a family of proteins that are thought to function as ATP-dependent RNA helicases. Pulse-chase analysis of ribosomal RNA synthesis and processing indicates that the drs1 mutant accumulates the 27S precursor of the mature 25S rRNA. These results suggest that, as in pre-mRNA splicing, RNA helicase activities are involved in ribosomal RNA processing.

• Liljas A
• Comparative biochemistry and biophysics of ribosomal proteins.
• Int Rev Cytol. 1991; 124: 103-36

• Tollervey D, Hurt EC
• The role of small nucleolar ribonucleoproteins in ribosome synthesis.
• Mol Biol Rep. 1990; 14: 103-6

• Mattaj IW
• A selective review of RNA-protein interactions in eukaryotes.
• Mol Biol Rep. 1990; 14: 151-5

• Uchiumi T
• [Acidic phosphoproteins of eucaryotic ribosomes].
• Seikagaku. 1989; 61: 1376-80

• Gezelius C, Eriksson A
• Neoplastic disease in a medicolegal autopsy material. A retrospective study in northern Sweden.
• Z Rechtsmed. 1988; 101: 115-30
• Display abstract

• Only a small fraction of sudden unexpected deaths are caused by neoplastic disease and thus subject ot medicolegal autopsy. The medicolegal autopsy forms an opportunity to study not only medically diagnosed and treated neoplasms, but also the natural evolution of untreated disease. In a series of 7,020 consecutive medicolegal autopsies in northern Sweden, we found 171 cases with malignant and/or intracranial neoplasms. In 41 cases, sudden death was caused by previously unknown tumors. The most common mechanisms of death in this group were disseminated cancer, intracranial tumors, pulmonary thromboembolism, hemoptysis, and aspiration of blood, and the most common locations were the bronchi and the lung. In some of these cases, the mechanism was sometimes dramatic, raising a question of violent death or intoxication. In 30 cases, sudden unexpected death was caused by previously known tumors, and also in this group disseminated cancer was the most common cause of death, and the most common locations were the bronchi and the lung. In 22 cases, tumors were found suicidal cases; in 14 of these, the tumor was considered to be a major causative factor to the suicide, while in eight cases the tumor was considered to be an incidental finding. The expected number of cancers in the 1,060 suicides investigated in this series was 27, according to the official cancer prevalence data. Thus, a possible over-representation of suicides among persons with cancer seems doubtful and needs further exploration.

• Boehm S
• A 5 S rRNA-like secondary structure in the 7 SL RNA may define a ribosomal binding site of the signal recognition particle.
• FEBS Lett. 1987; 212: 15-20
• Display abstract

• A new secondary structure model for parts of the 7 SL RNA is proposed which indicates for a stretch of at least 40 bases a strong structural homology to the ribosomal protein L5 binding site of eukaryotic 5 S rRNA. It is suggested that the 5 S rRNA-like structural part of 7 SL RNA mediates binding of the signal recognition particle near to the peptidyl transferase center of the ribosome.

• Nazar RN
• Evolutionary relationship between eukaryotic 29--32 S nucleolar rRNA precursors and the prokaryotic 23 S rRNA.
• FEBS Lett. 1982; 143: 161-2

• Loo MW, Schricker NS, Russell PJ
• Heat-sensitive mutant strain of Neurospora crassa, 4M(t), conditionally defective in 25S ribosomal ribonucleic acid production.
• Mol Cell Biol. 1981; 1: 199-207
• Display abstract

• A heat-sensitive mutant strain of Neurospora crassa, 4M(t), was studied in an attempt to define its molecular lesion. The mutant strain is inhibited in conidial germination and mycelial extension at the nonpermissive temperature (37 degrees C). Macromolecular synthesis studies showed that both ribonucleic acid (RNA) and protein syntheses are inhibited when 4-h cultures are shifted from 20 to 37 degrees C. Density gradient analysis of ribosomal subunits made at 37 degrees C indicated that strain 4M(t) is deficient in the accumulation of 60S ribosomal subunits in that the ratio of 60S/37S subunits was 0.29:1 compared with 1.6:1 for the parental strain. This phenotype was shown to be the result of a slow rate of processing of, and a deficiency in the amount of, the immediate precursor to 25S ribosomal RNA (the large RNA of the 60S subunit) in the sequence of events constituting the production of mature ribosomal RNAs from the primary transcript of the ribosomal deoxyribonucleic acid, the precursor ribosomal RNA molecule. Analysis of polysomes suggested that the heat-sensitive gene product might function in both the assembly and the function of the 60S ribosomal subunit, since there was a smaller proportion of newly made 60S subunits synthesized at 37 degrees C in the polysome region of the gradients than in the monosome-plus-subunit region. The ribosomal RNA processing defect is apparently responsible for the observed defects in germination and macromolecular synthesis at 37 degrees C, but the precise molecular lesion is not known. On the basis of these results, the heat-sensitive mutant allele in the 4M(t) strain is considered to define the rip1 (ribosome production) gene locus.

• Holmgren P, Lindquist O
• Lethal intoxications with morphine in Sweden 1966-1974.
• Z Rechtsmed. 1977; 79: 149-58
• Display abstract

• Fatal intoxications with morphine derivatives have become increasingly common in Sweden. Toxicologic data and pathologic findings in 34 cases of morphine intoxications from 1966 to 1974 in Sweden are presented. From 1972 on when morphine the black market, lethal intoxications with centrally stimulation amines.

• Breitenecker L
• [Clarification of accidents only through autopsy].
• Lebensversicher Med. 1976; 28: 90-5

• Greene MH
• Letter: Amphetamine death?
• JAMA. 1974; 227: 1259-1259

• Gunne LM, Holmgren P, Lindquist O, Saldeen T
• [Amphetamine poisoning].
• Lakartidningen. 1972; 69: 4373-6

• Orrenius S, Maehly AC
• Lethal amphetamine intoxication. A report of three cases.
• Z Rechtsmed. 1970; 67: 184-9

• Bruening G, Bock RM
• Covalent integrity and molecular weights of yeast ribosomal ribonucleic acid components.
• Biochim Biophys Acta. 1967; 149: 377-86

• Klein HP, Volkmann CM, Chao FC
• Fatty acid synthetase of Saccharomyces cerevisiae.
• J Bacteriol. 1967; 93: 1966-71
• Display abstract

• A light particle fraction of Saccharomyces cerevisiae, obtained from the crude ribosomal material, and containing the fatty acid synthetase, consisted primarily of 27S and 47S components. This fraction has a protein-ribonucleic acid ratio of about 13. Electron micrographs showed particles ranging in diameter between 100 and 300 A in this material. By use of density gradient analysis, the fatty acid synthetase was found in the 47S component. This component contained particles which were predominantly 300 A in diameter and which were considerably flatter than ribosomes, and it consisted almost entirely of protein.

• de Kloet SR, Strijkert PJ
• Selective inhibition of ribosomal RNA synthesis in Saccharomyces carlsbergensis by 5-fluorouracil.
• Biochem Biophys Res Commun. 1966; 23: 49-55

• FLETCHER TF
• Acute dextroamphetamine sulfate poisoning, report of a case in a child.
• AMA Am J Dis Child. 1953; 86: 777-9

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