In this application we propose to continue our study of molecular mechanisms in RNA splicing in intact cells using the novel MuSVts110 retrovirus as a model system. As outlined in this application, our previous work has led to an understanding of the role of intron sequences in specifying the commitment of this pre-mRNA to splice and the unique thermosensitivity of the splice event has allowed a promising beginning to the study of molecular mechanisms and interactions occurring during splicing in intact cells. We plan to extend these studies as follows: To facilitate the study of pre-mRNA/spliceosome component interactions in vivo, we plan to construct an MuSVts110 DNA whose transcription can be controlled through the replacement of the viral enhancer with an inducible enhancer, thus allowing us to follow preformed pre-mRNA through the splicing pathway without interference from nascent transcripts, or to study the splicing of nascent viral transcripts only. Interactions between viral transcripts and spliceosome components under splicing-permissive and nonpermissive conditions will be monitored through the use of antibodies specific for spliceosome components aided by techniques that allow crosslinking of pre-mRNAs and nucleic acid moieties of spliceosome components. We plan to continue studies on RNA structures that specify thermosensitive splicing. These studies will focus on the possibility that branchpoint positioning relative to 3' splice sites may help to regulate MuSVts110 splicing and the inference that specific exon structure may also be important. In this connection, we will also determine the nature of the changes made in the viral DNA of a series of 6m2 cell """"""""revertants"""""""" exhibiting stable relaxation in the growth temperature dependence of viral RNA splicing through amplification and sequencing using the PCR technique. Chimeric viruses will be constructed to distinguish splicing-critical versus neutral mutations.
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