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Cwc21p promotes the second step conformation of the spliceosome and modulates 3' splice site selection.

Gautam A, Grainger RJ, Vilardell J, Barrass JD, Beggs JD - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3BF, UK.

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Genetic interactions of cwc21Δ with U5 snRNA loop I alleles. (A) Diagram of yeast U5 snRNA loop I. Three U5 alleles are aligned below the WT sequence with alternative nucleotides highlighted in bold. The U5 loop I interaction with the exons before and after step I of splicing (according to (41)) are shown to the right and dashed lines represent known crosslinks. (B) Mutations in U5 snRNA loop I show synthetic lethality in the absence of CWC21. (C) Mutations in U5 snRNA loop I show temperature-sensitive defects in the absence of CWC21.
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Figure 2: Genetic interactions of cwc21Δ with U5 snRNA loop I alleles. (A) Diagram of yeast U5 snRNA loop I. Three U5 alleles are aligned below the WT sequence with alternative nucleotides highlighted in bold. The U5 loop I interaction with the exons before and after step I of splicing (according to (41)) are shown to the right and dashed lines represent known crosslinks. (B) Mutations in U5 snRNA loop I show synthetic lethality in the absence of CWC21. (C) Mutations in U5 snRNA loop I show temperature-sensitive defects in the absence of CWC21.

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.


Cwc21p promotes the second step conformation of the spliceosome and modulates 3' splice site selection.

Gautam A, Grainger RJ, Vilardell J, Barrass JD, Beggs JD - Nucleic Acids Res. (2015)

Genetic interactions of cwc21Δ with U5 snRNA loop I alleles. (A) Diagram of yeast U5 snRNA loop I. Three U5 alleles are aligned below the WT sequence with alternative nucleotides highlighted in bold. The U5 loop I interaction with the exons before and after step I of splicing (according to (41)) are shown to the right and dashed lines represent known crosslinks. (B) Mutations in U5 snRNA loop I show synthetic lethality in the absence of CWC21. (C) Mutations in U5 snRNA loop I show temperature-sensitive defects in the absence of CWC21.
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Figure 2: Genetic interactions of cwc21Δ with U5 snRNA loop I alleles. (A) Diagram of yeast U5 snRNA loop I. Three U5 alleles are aligned below the WT sequence with alternative nucleotides highlighted in bold. The U5 loop I interaction with the exons before and after step I of splicing (according to (41)) are shown to the right and dashed lines represent known crosslinks. (B) Mutations in U5 snRNA loop I show synthetic lethality in the absence of CWC21. (C) Mutations in U5 snRNA loop I show temperature-sensitive defects in the absence of CWC21.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3BF, UK.

Show MeSH
Related in: MedlinePlus