scholarly journals Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching

2018 ◽  
Author(s):  
Ruixue Wan ◽  
Rui Bai ◽  
Chuangye Yan ◽  
Jianlin Lei ◽  
Yigong Shi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Compelling Evidence ◽  
U2 Snrna ◽  
Branch Point Sequence ◽  
Catalytic Metal ◽  
Activated Complex

SummaryPre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the branching reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 Å away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 Å away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.

RNA Splicing by the Spliceosome

2020 ◽  
Vol 89 (1) ◽  
pp. 359-388 ◽  
Author(s):  
Max E. Wilkinson ◽  
Clément Charenton ◽  
Kiyoshi Nagai
Keyword(s):  
Splice Site ◽  
Active Site ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Structural Studies ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Mature Mrna ◽  
Catalytic Metal ◽  
Small Nuclear Ribonucleoproteins

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5′ splice site (5′SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5′SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5′SS, producing a free 5′ exon. Removal of the BP adenosine from the active site allows the 3′SS to bind, so that the 5′ exon attacks the 3′SS to produce mature mRNA and an excised lariat intron.

scholarly journals Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing

1999 ◽  
Vol 19 (8) ◽  
pp. 5352-5362 ◽  
Author(s):  
W. Hayes McDonald ◽  
Ryoma Ohi ◽  
Natalia Smelkova ◽  
David Frendewey ◽  
Kathleen L. Gould
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fission Yeast ◽  
Genetic Interaction ◽  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Immunoaffinity Purification ◽  
Snrnp Protein

ABSTRACT Myb-related cdc5p is required for G2/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiaesplicing factor Prp19p. cwf1 + is allelic to theprp5 + gene defined by the S. pombesplicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 andprp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lackingcdc5 + function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.

scholarly journals Characterization of nucleolin K88 acetylation defines a new pool of nucleolin colocalizing with pre-mRNA splicing factors

FEBS Letters ◽  
2013 ◽  
Vol 587 (5) ◽  
pp. 417-424 ◽  
Author(s):  
Sadhan Das ◽  
Rong Cong ◽  
Jayasha Shandilya ◽  
Parijat Senapati ◽  
Benoit Moindrot ◽  
...  
Keyword(s):  
Mrna Splicing ◽  
Splicing Factors

scholarly journals Synthetic Lethality of Yeast slt Mutations with U2 Small Nuclear RNA Mutations Suggests Functional Interactions between U2 and U5 snRNPs That Are Important for Both Steps of Pre-mRNA Splicing

1998 ◽  
Vol 18 (4) ◽  
pp. 2055-2066 ◽  
Author(s):  
Deming Xu ◽  
Deborah J. Field ◽  
Shou-Jiang Tang ◽  
Arnaud Moris ◽  
Brian P. Bobechko ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Two Factors ◽  
Synthetically Lethal ◽  
U5 Snrna ◽  
New Alleles

ABSTRACT A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5′ end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor andSLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709–713, 1996). The remaining four sltmutations are new alleles of previously identified splicing genes:slt15, previously identified as prp17(slt15/prp17-100), slt16/smd3-1,slt17/slu7-100, and slt21/prp8-21. slt11-1 andslt22-1 are synthetically lethal with mutations in the 3′ end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however,slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, includingslt15/prp17-100, slt17/slu7-100, andprp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179–5205, 1992), as well as with slt11-1, slt15/prp17-100,slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.

scholarly journals SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

1998 ◽  
Vol 140 (4) ◽  
pp. 737-750 ◽  
Author(s):  
Huan-You Wang ◽  
Wen Lin ◽  
Jacqueline A. Dyck ◽  
Joanne M. Yeakley ◽  
Zhou Songyang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Phosphorylation Sites ◽  
Sr Protein ◽  
Random Peptide ◽  
Selection For

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

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