Splicing defects in RNA export receptor mutants
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE93307 | GEO | 2018/01/01
SECONDARY ACCESSION(S): PRJNA360580
REPOSITORIES: GEO
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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 “hinge” that links the Prp8 Jab1-MPN regulatory “tail” 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
2016-07-21 | GSE75081 | GEO
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.
2016-07-21 | E-GEOD-75081 | biostudies-arrayexpress
Project description:Three shuttling SR-like proteins exist in Saccharomyces cerevisiae, Npl3, Gbp2 and Hrb1, that are involved in the nuclear export of mRNAs. In a screen for genes that regulate the export of Gbp2, we identified novel mutants of the splicing factors PRP8 and PRP17 that lead to severe mislocalization defects for Gbp2 and Hrb1, but not Npl3. Microarray and qRT-PCR analyses show that Gbp2 and Hrb1 preferentially bind to transcripts derived from intron-containing genes. Moreover, in contrast to Npl3, Gbp2 and Hrb1 show genetic and physical interactions with late splicing factors such as Prp17 and Prp43. Further, RNA co-immunoprecipitation experiments reveal that, unlike Npl3, association of Gbp2 and Hrb1 with the mRNA requires splicing, and this in turn is required for their Mex67 recruitment. We propose a model in which Gbp2 and Hrb1 are attached to the mRNAs at late stages of splicing to promote the subsequent export of spliced mRNAs. keyword: RIP-chip RNA-IP of endogenously expressed 3-fold C-terminal myc-tagged Gbp2 and Hrb1 in S288C background cells was each performed once in cells grown to a density of 4x10^7 cells/ml and hybridized against a total (input) RNA reference.
2013-08-30 | E-GEOD-20738 | biostudies-arrayexpress
Project description:The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss of function mutants in vivo. Here we show that inactivating the nonsense mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several splicing factors mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Î and prp18Î mutant strains reveals a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation. In addition, inactivation of Upf1 in the prp22-1 mutant enhances the unspliced precursor accumulation phenotype of several specific transcripts and partially suppresses growth defects associated with the prp17Î or prp22-1 mutations. These results support the idea that RNA surveillance by NMD mutes some of the effects of splicing factors mutations and show that the roles of splicing factors and their transcripts specificity cannot be fully understood in vivo unless RNA degradation systems that degrade unspliced precursors are also inactivated. Three samples from the Prp17delta, Prp18delta, Prp17deltaUpf1delta and Prp18Upf1delta mutants were grown indepedently and analyzed by tiling arrays to understand the role of the NMD component Upf1 in minimizing the accumulation of unspliced RNAs generated by the Prp17delta and Prp18delta splicing mutations.
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Project description:We performed RNA-seq to determine the impact of XAB2 depletion on global splicing. The results show that XAB2 depletion using two different siRNAs led to a wide range of splicing defects, with more than 50% events were intron retention.
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Project description:The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss of function mutants in vivo. Here we show that inactivating the nonsense mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several splicing factors mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Δ and prp18Δ mutant strains reveals a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation. In addition, inactivation of Upf1 in the prp22-1 mutant enhances the unspliced precursor accumulation phenotype of several specific transcripts and partially suppresses growth defects associated with the prp17Δ or prp22-1 mutations. These results support the idea that RNA surveillance by NMD mutes some of the effects of splicing factors mutations and show that the roles of splicing factors and their transcripts specificity cannot be fully understood in vivo unless RNA degradation systems that degrade unspliced precursors are also inactivated.
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