Heat treatment of nuclear extract alters selection of the 3' splice site in pre-mRNA splicing.

We investigated in vitro splicing reaction using an artificially created mRNA precursor containing a single 5' splice site and tandemly duplicated 3' splice sites. We found that the 3' splice site proximal to the 5' splice site is predominantly used under the standard splicing conditions. However, when the preheated nuclear extract was employed, the intermediate in which the distal 3' splice site was selected accumulated exclusively. This shows that heat treatment of nuclear extract abolishes the activity involved in the selection of the 3' splice sites that are in cis-competition for the common 5' splice site. The results presented here suggest the presence of a factor(s) required for the selection of the proximal 3' splice site.