Cwc21p promotes the second step conformation of the spliceosome and modulates 3' splice site selection.
Bottom Line: Here, we show that mutations in PRP16, PRP8, SNU114 and the U5 snRNA that affect this process interact genetically with CWC21, that encodes the yeast orthologue of the human SR protein, SRm300/SRRM2.Considering all the available data, we propose a role for Cwc21p positioning the 3' splice site at the transition to the second step conformation of the spliceosome, mediated through its interactions with the U5 snRNP.This suggests a mechanism whereby SRm300/SRRM2, might influence splice site selection in human cells.
Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3BF, UK.Show MeSH
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Mentions: Loop 1 of the U5 snRNA affects the alignment of exons in the spliceosome (21,41) (Figure 2A). We found that three U5 loop I mutations, U5-1, -2, -3 (Figure 2A), that affect the splicing of distinct subsets of pre-mRNAs and cause slow growth at 37°C (23,42) are synthetic lethal with deletion of CWC21 (Figure 2B). Also, the U5 loop I deletion mutants Δ94/95 (deletion of CC at positions 94 and 95) and Δ96/97 (deletion of UU at positions 96 and 97) that reduce the efficiency of step 2 by ∼50% (21), were lethal in conjunction with cwc21Δ, suggesting a role for Cwc21p in promoting step 2. The U5-ΔG93 and U5-Ins1U94/95 (insertion of an additional U between positions 94 and 95) alleles, although not lethal in combination with cwc21Δ, caused sensitivity to raised temperature, whereas there was no apparent effect with U5-Ins1U93/94 that is nearer one end of the loop (Figure 2C). These allele-specific effects are compatible with a role for Cwc21p helping to align or stabilize the exon substrates in the catalytic centre of the spliceosome.
Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3BF, UK.