A cis-acting selector of a 5' splice site. Cooperation between the sequence of the site and an upstream exonic element.

To investigate the mechanism by which a 5' splice site (D1) is selected, while a nearby potentially functional site (Dcr1) is silenced, we have studied the importance of the 9-nucleotide sequence of these 5' splice sites for their respective usage. Our model system uses a transcript derived from the early transcription unit 3 of adenovirus-2. Transcripts, harboring an exonic element previously shown to be required for the selection of D1 in the presence of Dcr1, were mutated in the D1 and Dcr1 sequences and assayed for splicing in vitro. We first show that an increased ability of D1 to pair with U1 small nuclear (sn) RNA correlates with an increased accumulation of splicing intermediates, independently of the presence of Dcr1. This variation of efficiency of the first splicing reaction does not significantly affect the overall splicing efficiency except when the potential D1-U1 snRNA hybrid is less than 6 base pairs. Equally, the selector activity of the upstream exon element requires a D1 sequence that is able to form hybrids of 6 base pairs or more with U1 snRNA. This indicates that the cis-acting selector of D1 includes the exonic element (a potential stem-loop structure) and a D1 sequence of sufficient strength.