We propose to continue using both genetic as well as biochemical methods for understanding the biogenesis and function of the snRNA components of mammalian nucleoplasmic and nucleolar snRNPs. All of the proposed experiments represent a continuation of work in progress using constructs, assay systems, and expertise which we have developed during the period of the previous award. Specifically, we propose (1) to continue using our human suppressor U1 snRNA (Zhuang and Weiner, 1986) for a second site reversion analysis of U1 structure and function (Yuo and Weiner, 1989); (2) to begin using our human suppressor U2 snRNA (Zhuang and Weiner, Genes Dev. in press) as the parental construct for an analogous second site reversion analysis of U2 snRNA structure and function; (3) to test possible U2/U6 base pairing interactions by asking whether mutations in a human suppressor U2 snRNA can be rescued by compensatory mutations in U6 snRNA; (4) to pursue our observation that the 5' end of rat U3 snRNA can be photocrosslinked in vivo to the 5' external transcribed spacer of the rRNA precursor (Stroke and Weiner, J. Mol. Biol. in press) by attempting to develop a suppressor U3 that can rescue processing of a mutant rRNA precursor; (5) to localize at the nucleotide sequence level the U2/U6 photocrosslink we have recently identified (Hausner, Giglio, and A.M.W., unpublished) and to characterize the step or steps in mRNA splicing at which this crosslink can be formed; (6) to use other crosslinking reagents and conditions to identify additional snRNA/snRNA and snRNA/mRNA interaction is between spliceosomal snRNPs; and (7) to test the hypothesis suggested by our earlier work (Hernandez and Weiner, 1986; Ach and Weiner, 1987) that the snRNA 3' end formation signal is an RNA polymerase II termination signal.
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