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Proteolysis of splicing factors during rat and monkey cell fractionation
H La Branche1, D Frappier, B Chabot
1Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
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.
Insights
Protease inhibitors improve nuclear extract activity for RNA splicing by preserving essential proteins like U2AF. SV40 T antigen transformation also enhances extract quality, suggesting reduced proteolysis in transformed cells.
Area of Science:
- Molecular Biology
- RNA Splicing Mechanisms
- Cellular Biochemistry
Background:
- Nuclear extracts are crucial for studying RNA splicing.
- Normal rat and monkey cell extracts often lack splicing activity.
- Reduced levels of splicing factors, such as U2AF, are observed in these extracts.
Purpose of the Study:
- To investigate the reasons behind the low splicing activity in normal cell extracts.
- To identify methods for improving the quality of nuclear extracts for splicing assays.
- To explore the role of proteolysis and viral transformation in extract preparation.
Main Methods:
- Preparation of nuclear extracts from various rat and monkey cell lines.
- Assessing splicing complex formation and U2AF binding activity.
- Utilizing protease inhibitors during cell fractionation.
- Analyzing extracts from cells transformed with viral antigens (SV40 T antigen, Py middle T antigen, v-fos).
Main Results:
- Normal FR3T3, rat-1, and CV-1 cell extracts showed poor splicing complex assembly and low U2AF binding.
- Protease inhibitors significantly increased U2AF and B protein levels, enhancing binding and complex formation.
- SV40 T antigen transformation yielded extracts capable of complex formation, with only SV40-transformed FR3T3 extracts showing splicing activity.
- Py middle T antigen transformation also improved extract quality, but v-fos transformation did not.
Conclusions:
- Proteolysis during nuclear extract preparation is a significant barrier to achieving splicing activity in normal rat and monkey cells.
- Viral T antigens appear to reduce proteolysis during cell fractionation, leading to more active extracts.
- These findings highlight the importance of controlling proteolysis for successful in vitro splicing studies.