NORMAL GENOMIC

• Eliana C, Javier E, Moises W
• Plasmodium falciparum spliceosomal RNAs: 3' and 5' end processing.
• Acta Trop. 2011; 117: 105-8
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• The major spliceosomal small nuclear ribonucleoproteins (snRNPs) consistof snRNA (U1, U2, U4 or U5) and several proteins which can be unique orcommon to each snRNP particle. The common proteins are known as Smproteins; they are crucial for RNP assembly and nuclear import ofspliceosomal RNPs. This paper reports detecting the interaction betweenPlasmodium falciparum snRNAs and Sm proteins, and the usual 5'trimethylated caps on the snRNAs, by immunoprecipitation with specificantibodies. Furthermore, an unusual poly(A) tail was detected on thesenon-coding RNAs.

• Palfi Z et al.
• SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.
• Genes Dev. 2009; 23: 1650-64
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• Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomescontain either the canonical heptameric Sm ring (U1, U5, spliced leadersnRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4snRNPs). Searching for specificity factors, we identified SMN and Gemin2proteins that are highly divergent from known orthologs. SMN issplicing-essential in trypanosomes and nuclear-localized, suggesting thatSm core assembly in trypanosomes is nuclear. We demonstrate in vitro thatSMN is sufficient to confer specificity of canonical Sm core assembly andto discriminate against binding to nonspecific RNA and to U2 and U4snRNAs. SMN interacts transiently with the SmD3B subcomplex, contactingspecifically SmB. SMN remains associated throughout the assembly of the Smheteroheptamer and dissociates only when a functional Sm site isincorporated. These data establish a novel role of SMN, mediating snRNPspecificity in Sm core assembly, and yield new biochemical insight intothe mechanism of SMN activity.

• Mesa A, Somarelli JA, Herrera RJ
• Spliceosomal immunophilins.
• FEBS Lett. 2008; 582: 2345-51
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• The spliceosome is a dynamic, macromolecular complex, which removesnon-protein-coding introns from pre-mRNA to form mature mRNA in a processknown as splicing. This ribonucleoprotein assembly is comprised of fiveuridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. Inhumans, several of the known proteinaceous splicing factors are members ofthe immunophilin superfamily. Immunophilins are peptidyl-prolyl cis-transisomerases that catalyze the conversion of proteins from cis to trans atXaa-Pro bonds. Our review of the data indicates that some members of thisprotein family are activators of spliceosomal proteins by way of foldingand transport.

• Liu Q, Liang XH, Uliel S, Belahcen M, Unger R, Michaeli S
• Identification and functional characterization of lsm proteins inTrypanosoma brucei.
• J Biol Chem. 2004; 279: 18210-9
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• RNA interference of Sm proteins in Trypanosoma brucei demonstrated thatthe stability of the small nuclear RNAs (U1, U2, U4, U5) and the splicedleader RNA, but not U6 RNA, were affected upon Sm depletion (Mandelboim,M., Barth, S., Biton, M., Liang, X. H., and Michaeli, S. (2003) J. Biol.Chem. 278, 51469-51478), suggesting that Lsm proteins that bind andstabilize U6 RNA in other eukaryotes should exist in trypanosomes. In thisstudy, we identified seven Lsm proteins (Lsm2p to Lsm8p) and examined thefunction of Lsm3p and Lsm8p by RNA interference silencing. Both Lsmproteins were found to be essential for U6 stability and mRNA decay.Silencing was lethal, and cis- and trans-splicing were inhibited.Importantly, silencing also affected the level of U4.U6 and the U4.U6/U5tri-small nuclear ribonucleoprotein complexes. The presence of Lsmproteins in trypanosomes that diverged early in the eukaryotic lineagesuggests that these proteins are highly conserved in both structure andfunction among eukaryotes. Interestingly, however, Lsm1p that is specificto the mRNA decay complex was not identified in the genome data base ofany kinetoplastidae, and the Lsm8p that in other eukaryotes exclusivelyfunctions in U6 stability was found to function in trypanosomes also inmRNA decay. These data therefore suggest that in trypanosomes only asingle Lsm complex may exist.

• Mura C, Cascio D, Sawaya MR, Eisenberg DS
• The crystal structure of a heptameric archaeal Sm protein: Implicationsfor the eukaryotic snRNP core.
• Proc Natl Acad Sci U S A. 2001; 98: 5532-7
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• Sm proteins form the core of small nuclear ribonucleoprotein particles(snRNPs), making them key components of several mRNA-processingassemblies, including the spliceosome. We report the 1.75-A crystalstructure of SmAP, an Sm-like archaeal protein that forms a heptamericring perforated by a cationic pore. In addition to providing directevidence for such an assembly in eukaryotic snRNPs, this structure (i)shows that SmAP homodimers are structurally similar to human Smheterodimers, (ii) supports a gene duplication model of Sm proteinevolution, and (iii) offers a model of SmAP bound to single-stranded RNA(ssRNA) that explains Sm binding-site specificity. The pronouncedelectrostatic asymmetry of the SmAP surface imparts directionality toputative SmAP-RNA interactions.

• Raker VA, Hartmuth K, Kastner B, Luhrmann R
• Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm siteRNA nonanucleotide in a specific and thermodynamically stable manner.
• Mol Cell Biol. 1999; 19: 6554-65
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• The association of Sm proteins with U small nuclear RNA (snRNA) requiresthe single-stranded Sm site (PuAU(4-6)GPu) but also is influenced bynonconserved flanking RNA structural elements. Here we demonstrate that anonameric Sm site RNA oligonucleotide sufficed for sequence-specificassembly of a minimal core ribonucleoprotein (RNP), which contained allseven Sm proteins. The minimal core RNP displayed several conservedbiochemical features of native U snRNP core particles, including a similarmorphology in electron micrographs. This minimal system allowed us tostudy in detail the RNA requirements for Sm protein-Sm site interactionsas well as the kinetics of core RNP assembly. In addition to the uridinebases, the 2' hydroxyl moieties were important for stable RNP formation,indicating that both the sugar backbone and the bases are intimatelyinvolved in RNA-protein interactions. Moreover, our data imply that aninitial phase of core RNP assembly is mediated by a high affinity of theSm proteins for the single-stranded uridine tract but that the presence ofthe conserved adenosine (PuAU.) is essential to commit the RNP particle tothermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Smsite oligonucleotide in core RNP assembly revealed that the regionsflanking the Sm site within the U snRNAs facilitate the kinetics of coreRNP assembly by increasing the rate of Sm protein association and bydecreasing the activation energy.

• Fury M, Andersen J, Ponda P, Aimes R, Zieve GW
• Thirteen anti-Sm monoclonal antibodies immunoprecipitate the threecytoplasmic snRNP core protein precursors in six distinct subsets.
• J Autoimmun. 1999; 12: 91-100
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• The small nuclear ribonucleoprotein particle (snRNP) common core proteinsare the lupus-associated Sm autoantigens. In mouse fibroblasts the sevensnRNP core proteins form a particle with a suggested stoichiometry ofB2[D1,D2(E,F,G)2] D3. Core particle assembly occurs in the cytoplasm wherenewly synthesized snRNAs assemble with core proteins stored in threeRNA-free complexes of (1) a 6S complex of [D1,D2(E,F,G)2] (2) a 20Scomplex of (B,D3 and an unidentified 70 kDa protein) and (3) a 2S-6Scomplex that minimally contains the B protein. In this report a panel of13 anti-Sm monoclonal antibodies is shown to immunoprecipitate sixdifferent subsets of the cytoplasmic snRNP proteins. Four epitopes areshared by the three aforementioned complexes and five other epitopes areshared by two of the complexes. In addition, the 6S or 20S complexes areapparently disrupted by five of the antibodies. Kinetic studies show thatthe three cytoplasmic snRNP protein complexes have independent half-lives.These studies provide another approach for characterizing the Sm epitopes.They also complement previous in vitro snRNP assembly studies and suggestthat snRNP core assembly occurs by the initial binding of snRNA to the 6Sparticle followed by addition of the B and D3 proteins.

• Bracken AP, Bond U
• Reassembly and protection of small nuclear ribonucleoprotein particles byheat shock proteins in yeast cells.
• RNA. 1999; 5: 1586-96
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• The process of mRNA splicing is sensitive to in vivo thermal inactivation,but can be protected by pretreatment of cells under conditions that induceheat-shock proteins (Hsps). This latter phenomenon is known as "splicingthermotolerance". In this article we demonstrate that the small nuclearribonucleoprotein particles (snRNPs) are in vivo targets of thermal damagewithin the splicing apparatus in heat-shocked yeast cells. Following aheat shock, levels of the tri-snRNP (U4/U6.U5), free U6 snRNP, and apre-U6 snRNP complex are dramatically reduced. In addition, we observemultiple alterations in U1, U2, U5, and U4/U6 snRNP profiles and theaccumulation of precursor forms of U4- and U6-containing snRNPs.Reassembly of snRNPs following a heat shock is correlated with therecovery of mRNA splicing and requires both Hsp104 and the Ssa Hsp70family of proteins. Furthermore, we correlate splicing thermotolerancewith the protection of a subset of snRNPs by Ssa proteins but not Hsp104,and show that Hsp70 directly associates with U4- and U6-containing snRNPsin splicing thermotolerant cells. In addition, our results show that Hsp70plays a role in snRNP assembly under normal physiological conditions.