Identification of a U2/U6 helix Ia mutant that influences 3 ' splice site selection during nuclear pre-mRNA splicing

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction b etween the U6 and U2 snRNAs, have previously been found to specifically blo ck the second catalytic step of nuclear pre-mRNA splicing. To further asses s the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivati ves, the spacing between the branch site adenosine and 3' splice site has b een reduced from 43 to 12 nt and this results in enhanced splicing of mutan ts in the conserved 3' terminal intron residue. In this context, mutation o f the conserved 3' intron terminal G to a C also results in the partial act ivation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identifi ed a mutation in the U6 snRNA (U57A) that significantly increases the selec tion of the cryptic 3' splice site over the normal 3' splice site and augme nts its utilization relative to that observed with the wild-type U2 or U6 s nRNAs. In a previous study, we found that the same U6 mutation suppressed t he effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch sit e and 3' splice site recognition suggests that this nucleotide may particip ate in the formation of the active site(s) of the spliceosome.