The experiments described in this proposal are designed to provide insights into the mechanisms and regulation of transcription by RNA polymerase II (RNAP II). Studies are proposed to investigate properties of several of the key components of TFIID and related factors, including TBP, the TBP-like protein (TLP) and certain TBP associated factors (TAFs); to understand how RNAP II is affected by the peptidyl-prolyl isomerase Pin1; and to elucidate the mechanism by which TLS/FUS proteins interface the processes of transcription and splicing. The following three Specific Aims are proposed: 1. TBP, TAF9 and related factors. The unexpected discovery that DT40 cells heterozygous for TBP (TBPHet) display significant growth defects will be pursued. Why cdc25B phosphatase is specifically underexpressed will be investigated, as will the finding TLP mRNA levels are significantly down regulated. Additional properties of TBP and TLP will be examined by genetic complementation, and the DNA binding properties of TLP will be investigated. The TAF dependence of several endogenous and artificial promoters will be characterized. Genetic complementation will be used to study properties of the TAF HFM, and possible interactions between specific TAF HFM heterodimers and DNA will be examined. Properties of a TAF9 related protein, TAF9a, will be investigated. 2. Pin1 and its interaction with RNAP II. Studies indicating that Pin1 influences the phosphorylation and function of RNAP II will be pursued. An inducible Pinl-overexpressing cell line will be used to test the idea that hyperphosphorylation of RNAP I! is sufficient to halt RNAP II transcription elongation and bring about RNAP II release. The mechanism of Pinl-induced hyperphosphorylation of RNAP IIO, which generates a novel, M phase-specific isoform, called RNAP IIOO, will be examined. The possibility that RNAP IIOO is totally inactive will be tested by purifying it from M phase cells and measuring its transcription activity and ability to stimulate pre-mRNA splicing. An in vitro """"""""transcript abortion"""""""" assay will be developed to investigate the mechanism responsible for M-phase specific RNAP II premature termination. 3. Functions of TLS/FUS proteins. RNA binding properties of two of these proteins, TLS/FUS and EWS, will be examined, and their interaction with RNAP II characterized. An in vitro coupled transcription-splicing assay will be developed to determine how added TLS/FUS influences the rate and/or extent of transcription and splicing. How these proteins function in vivo will be studied by first examining the association of splicing factors with elongating RNAP II. Cell lines over- or under-expressing TLS/FUS will then be developed and used to investigate how TLS/FUS affects the ability of splicing factors to associate with RNAP II, and how it influences transcription and splicing of target genes.
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