Js. Chang et Ds. Mcpheeters, Identification of a U2/U6 helix Ia mutant that influences 3 ' splice site selection during nuclear pre-mRNA splicing, RNA, 6(8), 2000, pp. 1120-1130
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.