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Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.

ABSTRACT: The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.

SUBMITTER: Chua K

PROVIDER: S-EPMC316594 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.

Chua K K Reed R R

Genes & development 19990401 7

The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the c ...[more]

PMID: 10197984

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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

Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.

Publications

Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.

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