Proteolysis of splicing factors during rat and monkey cell fractionation.

We have investigated the ability of various rat and monkey cell lines to yield nuclear extracts that would allow splicing of a model adenovirus pre-mRNA substrate. Extracts from normal FR3T3, rat-1 and CV-1 fibroblasts were unable to assemble splicing complexes and displayed a dramatic reduction in the binding activity of the splicing factor 65 kD U2AF. These results correlated with reduced levels of 65 kD U2AF and the snRNP-associated B protein. When a battery of protease inhibitors was used during cell fractionation, increased levels of 65 kD U2AF and B proteins were detected. Most importantly, U2AF binding and complex formation were dramatically improved in FR3T3, rat-1 and CV-1 extracts. Interestingly, transformation of rat and monkey cells with the SV40 large T antigen yielded extracts active in complex formation. Similar extracts were generated following transformation of rat-1 cells with the Py middle T antigen but not with the v-fos oncogene. Only SV40-transformed FR3T3 extracts displayed splicing activity. Our results indicate that proteolysis is a major obstacle encountered during the preparation of active extracts from normal rat and monkey cells and suggest that cells transformed with T antigens manifest reduced proteolysis during fractionation.