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 c ritical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We h ave identified prp8 mutants with the remarkable property of suppressing exo n ligation defects due to mutations in position 2 of the 5' GU and all posi tions of the 3' YAG. The prp8 mutants also suppress mutations in position A 51 of the critical ACAGAG motif in U6 snRNA which has been observed previou sly 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 fr om yeast to man, and from U2- to U12-dependent spliceosomes. We propose tha t Prp8 participates in a previously unrecognized tertiary interaction betwe en 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, arid assigns a fundamental role for Prp8 in pre-mRNA splicing.