The objective of this grant is to identify the molecular defect responsible for the temperature-sensitive RNA splicing phenotype of the ts110 Moloney murine sarcoma virus (Mo-MuSV). The viral genome (4.0 kb viral RNA) has suitable splice donor and acceptor sites that are used to generate a 3.5 kb viral mRNA which is translated to produce the transforming protein termed P85/gag- mos. This splicing reaction occurs readily at 28 degrees C in cells but not at 39 degrees C. There are two possible explanations for this viral conditional defect: First, the 4.0 kb viral RNA may have a structural alteration that does not allow itself to be spliced at restrictive temperatures; second, a heat-labile viral protein may participate in the splicing reaction; at restrictive temperatures it might block the splicing reaction. The following specific aims will be addressed to shed light on these two possibilities: 1) characterize viral RNA-containing nuclear complexes (spliceosomes) present in extracts of cells infected with ts110, its wild-type revertant and parental MuSV-124; 2) test ability of these spliceosome complexes to allow splicing; 3) construct MuSV DNA structures containing ts110 splicing elements capable of generating -600 nt 32/P-labeled pre-mRNAs in vitro with SP6 polymerase; and 4) test such pre-mRNAs for splicing in vitro; 5) analyze the structure of 32/P-labeled pre- mRNA intermediates and products in such a splicing system; 6) determine whether their splicing is ts in vitro; 7) if length intron in order to confer ts splicing phenotype; 8) study in vitro splicing with labeled chimeric ts110-adenovirus late pre-mRNA constructs to identify sequence elements involved in the conditional splicing defect; 9) Investigate the role of viral gene products in the splicing of 32/P-labeled pre-mRNA in nuclear extracts prepared from ts110 infected cells maintained at permissive and restrictive temperatures; 10) Test in vivo splicing by using full length ts110- like DNA, but with a short intron.
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