Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA183015 | ENA |
REPOSITORIES: ENA
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Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect. Splicing specific microarrays were used to assess suppression of the prp28-1 splicing defect by a prp8 allele (prp8-tes) that suppresses prp28-1 cold-sensitivity
2014-01-01 | E-GEOD-42754 | biostudies-arrayexpress
Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect.
2014-01-01 | GSE42754 | GEO
Project description:Prp8 Retinitis Pigmentosa Mutants Cause Defects in the Transition between the Catalytic Steps of Splicing
| PRJNA302382 | ENA
Project description:A structural “toggle” in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency
| PRJNA380015 | ENA
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:The spliceosome undergoes extensive rearrangements as it assembles in multiple steps onto the precursor messenger RNA. In the earliest assembly step, U1snRNA identifies the 5' splice site through base-pairing interactions. However, U1snRNA leaves the spliceosome relatively early in the assembly process. The 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components of the spliceosome during the complex assembly and rearrangements that build the catalytic site. Using a forward genetic screen in C. elegans, we have identified splicing suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new extragenic suppressor alleles from this screen, two in PRP8 and one in SNRNP200/Brr2. mRNASeq studies of these suppressor strains indicates that there are specific native targets with alternative 5' and alternative 3' splicing events affected by these suppressors, especially for the suppressor PRP8 D 1549N (position D1556 in humans). The strong suppressor at the unstructured N-terminus of SNRP200, N18K, indicates a potential regulatory role for this region. By examining distinct changes in the splicing of native genes, and by mapping these conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions in the spliceosome that are required to ensure that the initial 5'ss identified by U1snRNA early in spliceosome assembly is the one that gets loaded into the catalytic core.The spliceosome undergoes extensive rearrangements as it assembles in multiple steps onto the precursor messenger RNA. In the earliest assembly step, U1snRNA identifies the 5' splice site through base-pairing interactions. However, U1snRNA leaves the spliceosome relatively early in the assembly process. The 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components of the spliceosome during the complex assembly and rearrangements that build the catalytic site. Using a forward genetic screen in C. elegans, we have identified splicing suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new extragenic suppressor alleles from this screen, two in PRP8 and one in SNRNP200/Brr2. mRNASeq studies of these suppressor strains indicates that there are specific native targets with alternative 5' and alternative 3' splicing events affected by these suppressors, especially for the suppressor PRP8 D 1549N (position D1556 in humans). The strong suppressor at the unstructured N-terminus of SNRP200, N18K, indicates a potential regulatory role for this region. By examining distinct changes in the splicing of native genes, and by mapping these conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions in the spliceosome that are required to ensure that the initial 5'ss identified by U1snRNA early in spliceosome assembly is the one that gets loaded into the catalytic core.
2021-11-23 | GSE189437 | GEO
Project description:Aneuploidy and aging are correlated; however, a causal link between these two phenomena has remained elusive. Here we show that yeast disomic for a single native yeast chromosome generally have a decreased replicative lifespan. In addition, the extent of this lifespan deficit correlates with the size of the extra chromosome. We identified a mutation in BUL1 that rescues both the lifespan deficit and a protein trafficking defect in yeast disomic for chromosome 5. Bul1 is an E4 ubiquitin ligase adaptor involved in a protein quality-control pathway that targets membrane proteins for endocytosis and destruction in the lysosomal vacuole thereby maintaining protein homeostasis. Concurrent suppression of the aging and trafficking phenotypes suggests that disrupted membrane protein homeostasis in aneuploid yeast may contribute to their accelerated aging. The data reported here demonstrate that aneuploidy can impair protein homeostasis, shorten lifespan, and may contribute to age-associated phenotypes.
2015-12-04 | GSE75623 | GEO
Project description:Aneuploidy and aging are correlated; however, a causal link between these two phenomena has remained elusive. Here we show that yeast disomic for a single native yeast chromosome generally have a decreased replicative lifespan. In addition, the extent of this lifespan deficit correlates with the size of the extra chromosome. We identified a mutation in BUL1 that rescues both the lifespan deficit and a protein trafficking defect in yeast disomic for chromosome 5. Bul1 is an E4 ubiquitin ligase adaptor involved in a protein quality-control pathway that targets membrane proteins for endocytosis and destruction in the lysosomal vacuole thereby maintaining protein homeostasis. Concurrent suppression of the aging and trafficking phenotypes suggests that disrupted membrane protein homeostasis in aneuploid yeast may contribute to their accelerated aging. The data reported here demonstrate that aneuploidy can impair protein homeostasis, shorten lifespan, and may contribute to age-associated phenotypes. These are all CGH arrays comparing DNA content between the indicated strain of interest and a wt control.
2015-12-04 | E-GEOD-75623 | biostudies-arrayexpress