Sequences involved in the control of adenovirus L1 alternative RNA splicing.

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.