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Biogenesis of spliceosomal small nuclear ribonucleoproteins

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Abstract Virtually, all eukaryotic mRNAs are synthesized as precursor molecules that need to be extensively processed in order to serve as a blueprint for proteins. The three most prevalent processing steps are the capping reaction at the 5′‐end, the removal of intervening sequences by splicing, and the formation of poly (A)‐tails at the 3′‐end of the message by polyadenylation. A large number of proteins and small nuclear ribonucleoprotein complexes (snRNPs) interact with the mRNA and enable the different maturation steps. This chapter focuses on the biogenesis of snRNPs, the major components of the pre‐mRNA splicing machinery (spliceosome). A large body of evidence has revealed an intricate and segmented pathway for the formation of snRNPs that involves nucleo‐cytoplasmic transport events and elaborates assembly strategies. We summarize the knowledge about the different steps with an emphasis on trans‐acting factors of snRNP maturation of higher eukaryotes. WIREs RNA 2011 2 718–731 DOI: 10.1002/wrna.87 This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Processing > Splicing Mechanisms RNA Export and Localization > Nuclear Export/Import RNA in Disease and Development > RNA in Disease

Genomic structure and transcription factors of small nuclear RNA (snRNA) genes. (a) Genomic structure of polymerase (pol) II (U1) and pol III (U6) snRNA genes. Both classes of genes harbor the cis‐acting promoter elements distal sequence element (DSE) and proximal sequence element (PSE). In addition to these common elements, pol II snRNA genes also contain a 3′‐box. In contrast, pol III snRNA genes contain a TATA‐box between PSE and the encoding region that is absent in pol II snRNA genes. (b) Preinitiation complex formation on pol II and pol III snRNA genes. Formation of pol II snRNA preinitiation complex is initiated upon binding of Oct1 and STAF to DSE. This allows the recruitment of snRNA activating protein complex (SNAPc) to PSE. The general transcription factors TFIIA, TFIIB, TFIIE, TFIIF, TBP, TAF100 and possibly also TFIIH then join the promotor and allow eventually recruitment of pol II. Initiation also requires the Integrator complex, which binds to phosphorylated C‐terminal domain (CTD) of pol II. The pol III snRNA gene transcription initiation involves binding of the transcription factors Oct1 and STAF to DSE and SNAPc to PSE elements. TATA‐box binding protein (TBP) and the general pol III transcription factor TFIIIB, consisting of Brf and Bdp1 complete formation of the preinitiation complex. (c) Elongation and termination of pol II and pol III snRNA genes. Upon recruitment of pol II to the promoter region, serines 2, 5 and 7 of the CTD heptapeptide repeat are phosphorylated by specific kinases. m7G‐capping of the nascent snRNA 5′‐end occurs cotranscriptionally. Once the elongating pol II has passed the 3′‐box, the nascent pre‐snRNA is cleaved by the Integrator complex 5′ to the sequence element. Transcription is terminated by dephosphorylation of the CTD, nucleolytic degradation of the remaining RNA transcript and/or by additional factors binding downstream of the 3′‐box. Upon binding of pol III to the initiation complex and started transcription, the 3′‐end is capped by the methylphosphate‐capping enzyme (MePCE). A polyuridine stretch acts as termination signal which causes dissociation of pol III and/or cleavage by an yet undefined factor.

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Nuclear import of small nuclear ribonucleoproteins (snRNPs) and final maturation. After 3′‐end trimming by an exo‐ribonuclease and hypermethylation of the m7G‐cap by Tgs1, Spn1 associated with importin β binds the 2,2,7‐trimethylguanosine (m3G) cap structure. The Sm core domain represents a second import signal. The SMN complex and/or a yet unknown additional factor allow binding to importin β. After nuclear import and localization in Cajal bodies (CBs) the import complex dissociates. SnRNP‐specific proteins join the core snRNP and nucleotides within the small nuclear RNA (snRNA) are modified by scaRNPs (Ψ = pseudouridylation, m = 2′‐O‐ribose methylation). Mature snRNPs can now engage with the splicing machinery.

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Cytoplasmic assembly of U1 small nuclear ribonucleoproteins (snRNP). In the early phase of U snRNP assembly Sm proteins (i.e., B/B′, D1 and D3) are symmetrically dimethylated on designated arginine residues by the PRMT5 and/or PRMT7 complex and form together with pICln two distinct Sm protein subcomplexes (termed 6S complex and pICln‐B‐D3, respectively). The Sm proteins preassembled in the pICln–Sm subcomplexes are then transferred onto the survival motor neuron (SMN) complex; in both instances pICln is displaced upon transfer. Recruitment of small nuclear RNA (snRNA) and transfer of Sm core proteins onto the Sm site completes the Sm core formation. The SMN complex can then engage in a new round of assembly after being reloaded with Sm proteins by the PRMT5 complex.

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Export of pre‐snRNAs. The pre‐export complex assembles in Cajal bodies (CBs) upon binding of CBC 20 and CBC 80 to the m7G‐cap and the recruitment of phosphorylated adaptor of export protein (PHAX) to the cap binding complex (CBC). CRM1 together with GTP‐bound Ran contacts phosphorylated PHAX in the nucleoplasm to form the export complex. After transport through the nuclear pore complex (NPC) Ran‐GTP is hydrolyzed to GDP by Ran GTPase‐activating protein, RanGAP1. This results in the dissociation of CRM1 from the export cargo. Subsequently, PHAX dephosphorylation dissociates the export complex and the small nuclear RNA (snRNA) is released into cytoplasm. P, orthophosphate.

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RNA Processing > Splicing Mechanisms
RNA in Disease and Development > RNA in Disease
RNA Export and Localization > Nuclear Export/Import
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

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