Contrasted cis-acting effects of downstream 5' splice sites on the splicing of a retained intron: the adenoviral E1A pre-mRNA model.

摘要

The adenoviral E1A pre-mRNA contains an upstream intron (the 216 nucleotide intron) which is spliced only weakly both in vivo and in vitro. We have chosen the E1A transcript as a model to analyse, in vitro, the role of downstream cis-elements involved in the alternative splicing of this retained intron. By using a series of constructs containing specific deletions, mutations and/or truncations, we show that the 13S 5' splice site, positioned 259 nucleotides downstream of the 216 nucleotide intron, is the main cis-element which activates the splicing of this intron. Our results establish the importance of a downstream 5' splice site for the activation of the 3' splice site, which is known to be suboptimal within this retained intron. Unexpectedly, the 12S 5' splice site, although positioned at an ideal distance (121 nucleotides) from the upstream intron, does not exhibit such a cis-acting effect. In contrast, its improvement to a consensus sequence may even result in a slight negative cis-acting effect in the presence of the 13S 5' splice site, which is the first observation of such a feature. We have shown that this unexpected behaviour is due, at least partly, to the unusual characteristics of the wild-type upstream intron, which requires a hairpin structure between the branch sites and the 3' splice site to reduce the operational distance between these two sites. Possible mechanisms involved in the contrasted cis-acting effects of the 13S and 12S 5' splice sites are discussed.