Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro

We have developed an in vitro splicing system using a HeLa cell nuclear extract that is highly active for the alternative splicing of the natural E1A transcripts. The efficiency of using the three alternative 5′ splice sites is strongly dependent on the ionic conditions in the reaction, and the simultaneous production of the 13S, 12S, and 9S mRNA species is observed only at appropriate salt concentrations. All the intermediate and final splicing products have been extensively characterized and it has been demonstrated that the same major branch site is used for all the alternative reactions. The ratio of 13S to 9S mRNAs formed is close to that observed in vivo early in infection, suggesting that most of the mechanisms giving rise to alternative splicing are preserved in vitro.