Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA302382 | ENA |
REPOSITORIES: ENA
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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:RNA Splicing Factor Retinitis Pigmentosa
| PRJNA417002 | ENA
Project description:Mutations in pre-mRNA processing factors (PRPFs) cause autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause retinal disease. We have generated transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical-basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene-editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.
2018-08-20 | PXD010821 | Pride
Project description:SNRNP200 mutations cause autosomal dominant retinitis pigmentosa
| PRJDB10935 | ENA
Project description:SNRNP200 mutations cause autosomal dominant retinitis pigmentosa
| PRJDB10936 | ENA
Project description:A Drosophila mutant for the splicing factor Prp31 was generated and characterized as a model for Retinitis pigmentosa 11. The transcriptome of the mutant was compared to the genetic control white.
2019-01-01 | GSE99665 | GEO
Project description:Retinitis pigmentosa (RP) represents a heterogeneous group of hereditary eye disorders characterized by a progressive loss of vision. Mutations in thirty different genes have been linked to the autosomal dominant form of RP (adRP), with nearly one-quarter of them encoding the core components of the spliceosome, a macromolecular machine that removes introns from nascent pre-mRNAs, generating mature transcripts. Here we establish the Drosophila melanogaster model for RP13 linked to the mutations in the most conserved protein of the spliceosome, PRPF8/Prp8. We demonstrate negative impact of the nine different RP-associated Prp8 mutant proteins on the developmental timing when expressed in the endocrine cells specialized to produce the major insect molting hormone. In the developing eye primordium, actively cycling cells rather than differentiated photoreceptors showed sensitivity to Prp8 malfunction. The overexpression of the two most toxic RP variants Prp8S>F and Prp8H>R induced apoptosis and disturbances of the adult eye morphology. While the affected tissue mounted the stress and cytoprotective response, the genetic programs underlying neuronal function were attenuated. Importantly, the expressivity and penetrance among the RP-Prp8 mutations differed and increased under prp8 heterozygosity.
2020-06-16 | GSE139361 | GEO
Project description:Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5′ splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.
2021-01-06 | GSE151273 | GEO