title: Proteolysis of splicing factors during rat and monkey cell fractionation.
abstract: 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.
authors.affiliation: Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
authors.forename: H
authors.initials: H
authors.lastname: La Branche
authors.forename: D
authors.initials: D
authors.lastname: Frappier
authors.forename: B
authors.initials: B
authors.lastname: Chabot
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D002458:Cell Fractionation; D002460:Cell Line; D002461:Cell Line, Transformed; D000882:Haplorhini; D006367:HeLa Cells; D009687:Nuclear Proteins / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D012261:Ribonucleoproteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
chemical_list: D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear