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

Differential ASF/SF2 activity in extracts from normal WI38 and transformed WI38VA13 cells.

The normal human fibroblast cell line WI38 and a transformed derivative, WI38VA13, differentially splice fibronectin pre-mRNA in vivo. As a first step to understand the molecular basis for this regulation of splicing, we examined the ability of WI38 and WI38VA13 nuclear extracts to splice model adenovirus and globin pre-mRNAs. Adenovirus RNA splicing was detected in WI38VA13 but not in WI38 extracts. Likewise, when supplemented with a HeLa post-nuclear supernatant (S100), human beta-globin RNA splicing was detected in WI38VA13 but not in WI38 extracts. The splicing defect in WI38 extracts was associated with a reduced ability to form splicing complexes and with a corresponding decrease in the interaction of U2 small nuclear ribonucleoprotein (snRNP) with the branchsite. These defects did not correlate with a decrease in 65 kD U2AF binding since equivalent U2AF level and activity were detected in WI38 and WI38VA13 extracts. Rather, WI38 extracts displayed reduced ASF/SF2 activity and contained a low level of 30 and 40 kD SR phosphoproteins. Moreover, addition of purified ASF/SF2 dramatically increased splicing complex formation in WI38 extracts. These results raise the possibility that variations in the level and activity of ASF/SF2 and other SR proteins play a role in the regulation of fibronectin splicing

Messenger-RNA-binding proteins and the messages they carry

From sites of transcription in the nucleus to the outreaches of the cytoplasm, messenger RNAs are associated with RNA-binding proteins. These proteins influence pre-mRNA processing as well as the transport, localization, translation and stability of mRNAs. Recent discoveries have shown that one group of these proteins marks exon-exon junctions and has a role in mRNA export. These proteins communicate crucial information to the translation machinery for the surveillance of nonsense mutations and for mRNA localization and translation.N