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

摘要

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays 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 (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c⁷AG), little effect on the second step was observed, suggesting that the 6-NH2 and N⁷ groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N¹ group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.