NORMAL GENOMIC

• Shcherbik N, Pestov DG
• Ubiquitin and ubiquitin-like proteins in the nucleolus: multitasking tools for a ribosome factory.
• Genes Cancer. 2010; 1: 681-689
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• Synthesis of new ribosomes is an essential process upregulated during cell growth and proliferation. Here, we review our current understanding of the role that ubiquitin and ubiquitin-like proteins (UBLs) play in ribosome biogenesis, with a focus on mammalian cells. One important function of the nuclear ubiquitin-proteasome system is to control the supply of ribosomal proteins for the assembly of new ribosomal subunits in the nucleolus. Mutations in ribosomal proteins or ribosome assembly factors, stress, and many anticancer drugs have been shown to disrupt normal ribosome biogenesis, triggering a p53-dependent response. We discuss how p53 can be activated by the aberrant ribosome formation, centering on the current models of the interaction between ribosomal proteins released from the nucleolus and the ubiquitin ligase Mdm2. Recent studies also revealed multiple ubiquitin- and UBL-conjugated forms of nucleolar proteins with largely unknown functions, indicating that many new details about the role of these modifications in the nucleolus await to be discovered.

• Timmermans MJ, Roelofs D, Marien J, van Straalen NM
• Revealing pancrustacean relationships: phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers.
• BMC Evol Biol. 2008; 8: 83-83
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• BACKGROUND: In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences. RESULTS: In total 48 ribosomal proteins were obtained for the collembolan Folsomia candida. These 48 sequences were aligned with sequence data on 35 other ecdysozoans. Each ribosomal protein gene was available for 25% to 86% of the taxa. However, the total sequence information was unequally distributed over the taxa and ranged between 4% and 100%. A concatenated dataset was constructed (5034 inferred amino acids in length), of which ~66% of the positions were filled. Phylogenetic tree reconstructions, using Maximum Likelihood, Maximum Parsimony, and Bayesian methods, resulted in a topology that supports monophyly of Hexapoda. CONCLUSION: Although ribosomal proteins in general may not evolve independently, they once more appear highly valuable for phylogenetic reconstruction. Our analyses clearly suggest that Hexapoda is monophyletic. This underpins the inconsistency between nuclear and mitochondrial datasets when analyzing pancrustacean relationships. Caution is needed when applying mitochondrial markers in deep phylogeny.

• Wu B et al.
• Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus.
• Protein Sci. 2008; 17: 589-96
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• The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X(10)_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded beta-sheet with a simple beta2beta1beta3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered beta2-beta3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition.

• Song WJ, Qin QW, Qiu J, Huang CH, Wang F, Hew CL
• Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis.
• J Virol. 2004; 78: 12576-90
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• Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products.

• Murata T, Yoshino Y, Morita N, Kaneda N
• Identification of nuclear import and export signals within the structure of the zinc finger protein TIS11.
• Biochem Biophys Res Commun. 2002; 293: 1242-7
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• TIS11, a member of the CCCH zinc finger protein family, functions as a positive transcriptional regulator. TIS11 was localized in both the cytoplasm and nucleus when transiently expressed in COS-7 cells. Upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of TIS11 was observed, indicating that TIS11 shuttles between the nucleus and the cytoplasm. By deletion studies using a green fluorescent protein fusion system, we have mapped a functional nuclear localization signal (NLS) to a region containing two tandem repeats of the zinc finger motif of TIS11. A site-directed mutagenesis analysis of TIS11 NLS has revealed the critical importance of two arginine residues (Arg127 and Arg131 in the rat TIS11). Furthermore, we demonstrated that the N-terminal Leu-rich region of TIS11 serves as an LMB-sensitive nuclear export signal (NES), indicating that TIS11 follows a CRM1-mediated export pathway. These results suggest that TIS11 is subject to constant nucleocytoplasmic shuttling due to its NLS and NES.

• Watanabe T
• Isolation of a cDNA encoding a homologue of ribosomal protein L26 in the decapod crustacean Penaeus japonicus.
• Mol Mar Biol Biotechnol. 1998; 7: 259-62
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• A complementary DNA (PjL26) containing an open reading frame for a protein of 144 amino acids was isolated from the decapod crustacean Penaeus japonicus. The conceptual PjL26 protein exhibits significant sequence similarities to ribosomal protein L26 in vertebrates. The level of the PjL26 messenger RNA in the tail fan did not change significantly during the molt cycle. This cDNA, therefore, may serve as a useful control probe in Northern analyses of stage-specific transcripts in P. japonicus and related crustacean species.

• Tranque P, Crossin KL, Cirelli C, Edelman GM, Mauro VP
• Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.
• Proc Natl Acad Sci U S A. 1996; 93: 3105-9
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• To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.

• Tsui SK, Lee SM, Fung KP, Waye MM, Lee CY
• Primary structures and sequence analysis of human ribosomal proteins L39 and S27.
• Biochem Mol Biol Int. 1996; 40: 611-6
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• We report here the primary structures and sequence analysis of the human ribosomal protein L39 (hRPL39) and S27 (hRPS27). The hRPL39 cDNA is 352 bp in size encoding a predicted protein of 51 amino acids. The region KRRHWRRTKL near the carboxyl end is conserved between human, rat, maize, C. elegans and yeast. The hRPS27 cDNA is 321 bp in size encoding a deduced protein of 84 amino acids. When the deduced amino acid sequence of hRPS27 was compared with that of rat ribosomal protein S27 and human metalloproteinstimulin-1 (MPS-1), identity levels of 96.4% and 100% were obtained respectively. A potential polyadenylation signal AACAAA is found in the MPS-1 cDNA but the more frequently used AATAAA sequence is present in the hRPS27 cDNA. The carboxyl-terminal cysteine arrangement in hRPS27 is similar to the family of C4 zinc finger DNA-binding proteins.

• Baker RT, Williamson NA, Wettenhall RE
• The yeast homolog of mammalian ribosomal protein S30 is expressed from a duplicated gene without a ubiquitin-like protein fusion sequence. Evolutionary implications.
• J Biol Chem. 1996; 271: 13549-55
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• In mammals, the 59-residue ribosomal protein S30 (rpS30) is synthesized as a fusion to a 74-residue ubiquitin-like protein, which is cleaved to yield mature rpS30. An artificial fusion of this ubiquitin-like protein to E. coli beta-galactosidase was not cleaved when expressed in yeast (Saccharomyces cerevisiae), indicating that yeast lack this cleaving activity. The yeast rpS30 homolog (yrpS30) was purified and sequenced to reveal a 63-residue protein with 61% sequence identity to mammalian rpS30. Degenerate oligonucleotides based on the yrpS30 sequence were used to isolate full-length yrpS30 cDNAs. Sequence analysis of five cDNA clones revealed that yrpS30 is not synthesized as a fusion to a ubiquitin-like protein but is extended at its N terminus by a single methionine residue. The corresponding gene was identified in the GenBankTM data base by sequence alignment and termed RPS30A. The gene consists of two exons separated by a 430-base pair intron, which contains consensus splicing elements. Exon 1 encodes the initiator methionine residue and is preceded by canonical yeast ribosomal protein gene promoter elements. Exon 2 encodes the 62-residue mature yrpS30. Genomic hybridization reveals that the RPS30A gene is duplicated. Disruption of the RPS30A gene is not lethal but confers a slow growth phenotype. Ribosomes in the mutant strains contain an authentic yrpS30 protein, indicating that a functional yrpS30 is expressed from the duplicated gene but that the reduced capacity for yrpS30 synthesis restricted the growth rate. Analysis of available DNA sequence data bases reveals that rpS30 is synthesized as a fusion to a ubiquitin-like protein in nematodes and mammals but unfused in yeast, plants, and protazoa.

• Chan YL, Wool IG
• The primary structure of rat ribosomal protein L22.
• Biochim Biophys Acta. 1995; 1260: 113-5
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• The amino acid sequence of the rat 60S ribosomal subunit protein L22 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L22 has 128 amino acids; the molecular weight is 14,779. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 6 to 9 copies of the L22 gene. The mRNA for the protein is about 600 nucleotides in length. Rat L22 is related to ribosomal proteins from other eukaryotes and is identical in amino acid sequence to human EAP, the EBER 1 (Epstein-Barr virus encoded RNA) associated protein.

• Tsiboli P, Choli T
• Studies on S14 protein from Thermus thermophilus possessing zinc finger-like motifs.
• Biol Chem Hoppe Seyler. 1995; 376: 127-30
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• The amino acid sequence of the ribosomal protein S14 of Thermus thermophilus has been determined both by automated sequence analysis of the intact protein as well as by DNA sequence analysis of the gene. The carboxy-terminal region was verified by both amino acid sequence analysis of the carboxy-terminal peptide produced after Glu-C digestion and by DNA sequence analysis. The protein contains 60 amino acid residues with a calculated molecular weight of 7008. The most extensive homology is observed in the carboxy-terminal regions of all S14 proteins compared. Interestingly, the carboxy-terminal region of most S14 proteins of all species studied so far, form zinc-finger domains in the variety of C2-C2 form.

• Chan YL, Olvera J, Paz V, Wool IG
• The primary structure of rat ribosomal protein S15a.
• Biochem Biophys Res Commun. 1994; 200: 1498-504
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• The amino acid sequence of the rat 40S ribosomal subunit protein S15a was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein S15a has 129 amino acids, the NH2-terminal methionine is removed after translation of the mRNA and has a molecular weight of 14,698. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 10 to 15 copies of the S15a gene. The mRNA for the protein is about 650 nucleotides in length. Rat S15a is related to ribosomal proteins from other eukaryotes, from eubacteria, and from archaebacteria.

• Wong JM et al.
• Ubiquitin-ribosomal protein S27a gene overexpressed in human colorectal carcinoma is an early growth response gene.
• Cancer Res. 1993; 53: 1916-20
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• One of the extension proteins on the carboxy terminus of ubiquitin was reported as the ribosomal protein S27a. We have cloned a gene which encodes this ubiquitin hybrid protein from a complementary DNA library of a human colon carcinoma cell line. Northern blot analysis of surgical specimens from colon cancer patients showed that these messenger RNA levels were higher in tumor tissue than in adjacent normal mucosa. Furthermore, to investigate the role of this novel ubiquitin hybrid gene in cellular growth control, the responsiveness of this gene to serum growth factors was examined. Within 30 min after serum or 12-O-tetradecanoylphorbol-13-acetate stimulation, its messenger RNA expression in rat fibroblast cells (Rat 1) was increased. Nuclear runoff transcription studies showed that the kinetics of induction of this gene is almost identical to that of protooncogene c-jun or c-fos, the known early growth response genes. Thus, this ubiquitin hybrid gene appears to be a novel early growth response gene overexpressed in human colon cancer and warrants further studies in the pathogenesis of colorectal carcinoma.

• Chan YL, Wool IG
• The primary structure of rat ribosomal protein L8.
• Biochem Biophys Res Commun. 1992; 185: 539-47
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• The amino acid sequence of the rat 60S ribosomal subunit protein L8 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L8 has 257 amino acids and has a molecular weight of 28,007. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 4 or 5 copies of the L8 gene. The mRNA for the protein is about 950 nucleotides in length. Rat L8 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.

• Chan YL, Wool IG
• The primary structure of rat ribosomal protein S25.
• Biochem Biophys Res Commun. 1992; 186: 1688-93
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• The amino acid sequence of the rat 40S ribosomal subunit protein S25 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein S25 has 125 amino acids and has a molecular weight of 13,733. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 19 to 22 copies of the S25 gene. The mRNA for the protein is about 550 nucleotides in length. Rat S25 is homologous to ribosomal proteins from other eukaryotes (human and yeast).

• Kuwano Y, Olvera J, Wool IG
• The primary structure of rat ribosomal protein L38.
• Biochem Biophys Res Commun. 1991; 175: 551-5
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• The amino acid sequence of the rat 60S ribosomal protein L38 was deduced from the sequence of nucleotides in three recombinant cDNAs. Ribosomal protein L38 has 69 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 8,081. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 11-13 copies of the L38 gene. The mRNA for the protein is about 450 nucleotides in length.

• Carol P, Li YF, Mache R
• Conservation and evolution of the nucleus-encoded and chloroplast-specific ribosomal proteins in pea and spinach.
• Gene. 1991; 103: 139-45
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• Two cDNA clones have been isolated from a lambda g11 cDNA library constructed with poly(A)+ mRNAs prepared from spinach seedlings. These nuclear cDNAs encode chloroplast (cp) ribosomal (r) proteins designated L24 and L40. These r-proteins have been identified in the cp 50S r-subunit by immunoblot analysis, amino acid (aa) composition and N-terminal aa sequencing. The L24 r-protein contains a central eubacterial homologous core with the N- and C-terminal polypeptide extensions. The L40 r-protein has no homologous counterpart in bacterial ribosomes. The two nuclear encoded r-proteins have their homologues in pea, a legume, showing that specific elements of cp ribosomes are conserved in higher plants. Surprisingly, the cp-specific r-protein L40 has a higher aa substitution rate than that of other eubacterial-like cp r-proteins identified previously in pea and spinach.

• Chan YL, Paz V, Wool IG
• The primary structure of rat ribosomal protein L23.
• Biochem Biophys Res Commun. 1991; 178: 1153-9
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• The amino acid sequence of the rat 60S ribosomal subunit protein L23 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L23 has 140 amino acids and a molecular weight of 14,856. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the L23 gene. The mRNA for the protein is about 600 nucleotides in length. Rat L23 is homologous to Saccharomyces cerevisiae L17a and related to Escherichia coli L14 and other members of the prokaryotic L14 family.

• Suzuki K, Olvera J, Wool IG
• The primary structure of rat ribosomal protein S13.
• Biochem Biophys Res Commun. 1990; 171: 519-24
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• The covalent structure of the rat 40S ribosomal subunit protein S13 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Rat S13 contains 150 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 17,080. Hybridization of a S13 cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the gene for the protein. The mRNA for the protein is about 620 nucleotides in length. Rat S13 is related to Saccharomyces cerevisiae YS15 and to Halobacterium marismortui S11. The protein contains a possible internal duplication of 12 residues.

• Suzuki K, Olvera J, Wool IG
• The primary structure of rat ribosomal protein L9.
• Gene. 1990; 93: 297-300
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• The amino acid (aa) sequence of rat ribosomal (r) protein L9 was deduced from the nucleotide (nt) sequence in a recombinant cDNA and confirmed from the N-terminal aa sequence of the protein. L9 contains 192 aa and has an Mr of 21879. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 20-23 copies of the L9 gene. The mRNA for the protein is about 800 nt in length. Rat L9 is related to Saccharomyces cerevisiae YL11, Methanococcus vannielii L6, Escherichia coli L6 and other members of the prokaryotic L6 family. The protein contains a possible internal duplication of 11 aa.

• Suzuki K, Olvera J, Wool IG
• The primary structure of rat ribosomal protein L35.
• Biochem Biophys Res Commun. 1990; 167: 1377-82
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• The amino acid sequence of the rat 60S ribosomal subunit protein L35 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein L35 has 122 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 14,412. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-17 copies of the L35 gene. The mRNA for the protein is about 570 nucleotides in length. Rat L35 is related to the archaebacterial ribosomal proteins Halobacterium marismortui L33 and Halobacterium halobium L29E; it is also related to Escherichia coli L29 and to other members of the prokaryotic ribosomal protein L29 family. The protein contains a possible internal duplication of 11 residues.

• Wool IG, Chan YL, Paz V, Olvera J
• The primary structure of rat ribosomal proteins: the amino acid sequences of L27a and L28 and corrections in the sequences of S4 and S12.
• Biochim Biophys Acta. 1990; 1050: 69-73
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• The amino acid sequences of rat ribosomal proteins L27a and L28 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed from the NH2-terminal amino acid sequences of the proteins. L27a contains 147 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 16 476. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 18-22 copies of the L27a gene. The mRNA for the protein is about 600 nucleotides in length. L27a is homologous to mouse L27a (there are 3 amino acid changes) and to yeast L29. Rat ribosomal protein L28 has 136 amino acids (its NH2-terminal methionine is also processed after translation) and has a molecular weight of 15 707. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 9 or 10 copies of the L28 gene. The mRNA for the protein is about 640 nucleotides in length. L28 contains a possible internal duplication of 9 residues. Corrections are recorded in the sequences reported before for rat ribosomal proteins S4 and S12.

• Suzuki K, Olvera J, Wool IG
• The primary structure of rat ribosomal protein S7.
• FEBS Lett. 1990; 271: 51-3
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• The amino acid sequence of the rat 40S ribosomal subunit protein S7 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the amino acid sequence of a cyanogen bromide peptide obtained from the protein. Ribosomal protein S7 has 194 amino acids and has a molecular mass of 22,113. Hybridization of the cDNA to digest of nuclear DNA suggests that there are 14-16 copies of the S7 gene. The mRNA for the protein is about 725 nucleotides in length. Rat S7 is homologous with Xenopus laevis S8. The protein contains a possible internal duplication of 10 residues.

• Aoyama Y, Chan YL, Meyuhas O, Wool IG
• The primary structure of rat ribosomal protein L18a.
• FEBS Lett. 1989; 247: 242-6
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• The amino acid sequence of rat ribosomal protein L18a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L18a contains 175 amino acids and has a molecular mass of 20,047 Da. Hybridization of the cDNA to digests of rat nuclear DNA and to a preparation of poly(A)+ mRNA suggests that there are 8-11 copies of the L18a gene and that the mRNA for the protein is about 700 nucleotides in length. Rat L18a is related to Schizosaccharomyces pombe L17 and perhaps to Halobacterium marismortui L19.