The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.