Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome.
The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing.
Collins
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Graduate Group in Biophysics, University of California San Francisco (UCSF), San Francisco, California 94143-0448, USA.
Guthrie
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D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence / Q000235:genetics; D005091:Exons / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D016153:Genes, Suppressor / Q000235:genetics; D007438:Introns / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012326:RNA Splicing / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
D005656:Fungal Proteins; C077018:PRP8 protein, S cerevisiae; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins