Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase?

The use in vivo of an alternative 5' splice site sequestered within a short stem of potential RNA secondary structure is determined by the length of the loop. Above a threshold length of loop, the alternative site is used despite the potential structure. In contrast, the alternative site is used very little or not at all during splicing in vitro with all lengths of loop that we have tested. A model is proposed which suggests that pre-mRNA is free to fold only within a limited period after transcription.