snRNA genes encode the RNA constituents of the U1 and U2 snRNPs, which are involved in mRNA splicing. They are thought to be transcribed by RNA polymerase II because synthesis of the U1 and U2 snRNA is sensitive to low concentrations of alpha- amanitin. However, these genes differ in several respects from mRNA-encoding genes; in particular, the 3' ends of snRNAs seem to be generated by a mechanism unique among polymerase II transcripts. U1 and U2 RNAs are first synthesized as precursors (pre-U1 and pre-U2) elongated by a few nucleotides (nt) at the 3' end. These precursors are then shortened to the mature size, probably by an exonuclease. Whether pre-U1 and pre-U2 RNAs are generated by termination of transcription or by processing of even longer RNA molecules is unclear. It is known, however, that the reaction requires a conserved sequence (the """"""""3' box"""""""") located downstream of the site of 3' end formation, as well as snRNA promoter elements. This suggests that the 3' end of snRNAs is formed either by termination of transcription or by a processing reaction that is coupled to transcription. We plan to study the mechanisms of expression of snRNA genes, and in particular the mechanism of 3' end formation of the RNAs. First, we will perform run-on transcription experiments in isolated nuclei to determine whether transcription from snRNA promoters terminates close to or at the site of pre-U1 3' end formation. If this were the case, U1 would be the first example of a polymerase II transcript whose mature 3' end is not generated by the endonucleolytic cleavage of a long precursor. Second, we will continue our studies to define which snRNA promoter elements are involved in 3' end formation of the RNAs; it will be of particular interest to find out whether elements necessary for initiation of transcription are physically distinct from elements required for 3' end formation. Third, we will attempt to reproduce accurate and efficient transcription of snRNA genes in a soluble cell extract. Indeed, there is now no system capable of accurately transcribing mammalian snRNA genes, and this has precluded the functional analysis of the factors involved in snRNA gene transcription. Fourth, will will determine whether the 3' box and the snRNA promoters, in particular the elements involved in 3' end formation, contain binding sites for specific proteins; we will map precisely these binding sites, and purify the binding proteins. The functional role of these proteins will then be examined using the in vitro transcription system. Together, these experiments will provide a detailed understanding of the mechanisms of snRNA gene transcription.
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