Role of adenine functional groups in the recognition of the 3 '-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3' splice sites of metazoan nuclear pre-mRNAs pl ays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (A G --> CG) inhibits this step. We find that the second step was even more se verely inhibited by cytosine (AC --> CG) or uracil (AC --> UG) substitution s at this position. By contrast, a relatively moderate inhibition was obser ved with a hypoxanthine substitution (AC --> HG). When adenine was replaced by a purine base (AC --> PG) or by 7-deazaadenine (AG --> c(7)AG), little effect on the second step was observed, suggesting that the 6-NH2 and N-7 g roups do not play a critical role in adenine recognition. Finally, replacem ent of adenine by 2-aminopurine (AG --> 2-APG) had no effect on the second step. Taken together, our results suggest that the N-1 group of adenine fun ctions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.