Transcription of U6 Small Nuclear RNA

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Human nuclear U6 RNA is presumably tanscribed by polymerase III unlike the other abundant snRNAs involved in pre-mRNA splicing. However the 5' flanking regions of U6 genes from a variety of organisms contain transcription control elements that appear to be very similar to those employed on pol II- transcribed U1 and U2 genes. Moreover, unlike typical pol III genes, no internal control elements have been found in the U6 snRNA gene. In one project a detailed analysis will be made of the transcription control elements in the proximal region of a human U6 gene. Experiments are proposed that use in vitro transcription and experession in vivo from transfected genes to investigate the promoter element(s) that cause the U6 gene to be transcribed by RNA polymerase III. The U6 "basal" promoter situated in the 5' proximal region appears to be rigidly constrained with respect to the relative spacing and/or orientation of control elements. The role of spacing will be studied in detail through the construction of a series of deletion and insertion mutants in the U6 gene promoter. The mechanism of U6 transcription will also be studied by partial purification of trans-acting factors. A S100 extract that supports accurate U6 transcription will fractionated through several chromatographic steps. Direct transcription assays and DNA- protein binding analyses such as DNase footprinting will be used to characterize the partially purified factors. The central focus of this application is the control of human U6 small nuclear RNA (snRNA) transcription. This snRNA is a cofactor in pre-messenger RNA splicing, a fundamental step of gene expression in eukaryotic cells.