Abstract
We have studied the effect of the 5' cap structure on the splicing of precursor mRNAs containing three exons and two introns within a single molecule in a HeLa nuclear extract. When a precursor mRNA was capped, the upstream intron was spliced out more efficiently than the downstream intron. The differential splicing reactions of the two introns are not due to differences in the intrinsic efficiency of splicing of each intron, since the preferential excision of the upstream intron was also observed when the positions of the two introns relative to the cap structure were reversed. When uncapped precursor mRNA was used as substrate, the downstream intron was spliced out appreciably, but splicing of the upstream intron was greatly reduced. Preincubation of the extract with cap analogues inhibited splicing of the upstream intron but not the downstream intron. Thus, the cap structure exerts its effect primarily on the 5' proximal intron.