Dataset Information

Yeast Prp2 liberates the 5' splice site and the branch site adenosine for catalysis of pre-mRNA splicing.

ABSTRACT: The RNA helicase Prp2 facilitates the remodeling of the spliceosomal Bact complex to the catalytically activated B* complex just before step one of splicing. As a high-resolution cryo-EM structure of the B* complex is currently lacking, the precise spliceosome remodeling events mediated by Prp2 remain poorly understood. To investigate the latter, we used chemical structure probing to compare the RNA structure of purified yeast Bact and B* complexes. Our studies reveal deviations from conventional RNA helices in the functionally important U6 snRNA internal stem-loop and U2/U6 helix Ib in the activated Bact complex, and to a lesser extent in B*. Interestingly, the N7 of U6-G60 of the catalytic triad becomes accessible to DMS modification in the B* complex, suggesting that the Hoogsteen interaction with U6-A52 is destabilized in B*. Our data show that Prp2 action does not unwind double-stranded RNA, but enhances the flexibility of the first step reactants, the pre-mRNA's 5' splice site and branch site adenosine. Prp2 therefore appears to act primarily as an RNPase to achieve catalytic activation by liberating the first step reactants in preparation for catalysis of the first step of splicing.

SUBMITTER: Bao P

PROVIDER: S-EPMC5688998 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Yeast Prp2 liberates the 5' splice site and the branch site adenosine for catalysis of pre-mRNA splicing.

Bao Penghui P Höbartner Claudia C Hartmuth Klaus K Lührmann Reinhard R

RNA (New York, N.Y.) 20170901 12

The RNA helicase Prp2 facilitates the remodeling of the spliceosomal B<sup>act</sup> complex to the catalytically activated B* complex just before step one of splicing. As a high-resolution cryo-EM structure of the B* complex is currently lacking, the precise spliceosome remodeling events mediated by Prp2 remain poorly understood. To investigate the latter, we used chemical structure probing to compare the RNA structure of purified yeast B<sup>act</sup> and B* complexes. Our studies reveal devia ...[more]

PMID: 28864812

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Project description:Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

Dataset Information

Yeast Prp2 liberates the 5' splice site and the branch site adenosine for catalysis of pre-mRNA splicing.

Publications

Yeast Prp2 liberates the 5' splice site and the branch site adenosine for catalysis of pre-mRNA splicing.

Similar Datasets

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