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Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

ABSTRACT: Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

SUBMITTER: Mayerle M

PROVIDER: S-EPMC5422793 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

Mayerle Megan M Raghavan Madhura M Ledoux Sarah S Price Argenta A Stepankiw Nicholas N Hadjivassiliou Haralambos H Moehle Erica A EA Mendoza Senén D SD Pleiss Jeffrey A JA Guthrie Christine C Abelson John J

Proceedings of the National Academy of Sciences of the United States of America 20170417 18

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that <i>prp8</i> alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, th ...[more]

PMID: 28416677

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Project description:The essential process of pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome employs DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appro-priate points in the splicing cycle and repressing it at others. Mutations linked to Retinitis Pig-mentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous In vitro studies of homologous mutations in Saccharomyces cerevisiae show that Prp8-RP mutants cause defects in spliceosome activation. Here we show a subset of RP muta-tions in Prp8 also cause defects in the transition between the 1st and 2nd catalytic steps of splic-ing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the 1st and 2nd catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splic-ing efficiency. The location of the Prp8-RP mutants, at the â??hingeâ?? that links the Prp8 Jab1-MPN regulatory â??tailâ?? to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2 activity. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing ca-talysis. paper to be submitted Two channel microarrays were used. RNA isolated from wt yeast grown simultaneously to the mutant was used as a reference. This reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each mutant relative to the wt. Three independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Strains, both WT and mutant, were grown at 37C until mid-log phase, OD600 of approximately 0.7. The mutated PRP8 gene is present on HIS marked CEN plasmid, and the corresponding genomic copy of PRP8 deleted. Strains labeled as wildtype also have the relevant genomic PRP8 deleted, but complemented with wildtype PRP8 on CEN plasmid.

Dataset Information

Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

Publications

Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

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