RNA polymerase III; Maf1; Brf2; transcription regulation; U6 snRNA gene; TORC1; genome
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James Faresse Nicole, Canella Donatella, Praz Viviane, Michaud Joëlle, Romascano David, Hernandez Nouria (2012), Genomic Study of RNA Polymerase II and III SNAP(c)-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators., in PLoS genetics
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Orioli A, Pascali C, Quartararo J, Diebel KW, Praz V, Romascano D, Percudani R, van Dyk LF, Hernandez N, Teichmann M, Dieci G (2011), Widespread occurrence of non-canonical transcription termination by human RNA polymerase III, in NUCLEIC ACIDS RESEARCH
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1. SUMMARY OF RESEARCH PLAN RNA polymerase III (pol III) transcribes various short genes encoding untranslated RNAs involved in protein biosynthesis and in the maturation of other RNA molecules. Pol III is recruited to three main types of promoters, types 1, 2, and 3. The type 3 promoters, exemplified by the human U6 small nuclear (sn) RNA promoter, are located upstream of the RNA coding sequence. Remarkably, they are very similar to the promoters of the U1 and U2 snRNA genes, which are recognized by pol II. The human snRNA promoters thus offer a unique system to study how a promoter and its associated transcription factors (TFs) achieve recruitment of a specific RNA polymerase, here pol II or pol III. Pol III transcription is highly regulated with cell growth and proliferation, and it is invariably up-regulated in tumors. Recent results show that up-regulation of pol III transcription leads to cell transformation, giving renewed interest in mechanisms of pol III transcription regulation. The single-subunit RNAP-IVSP is expressed from an alternatively spliced transcript of the nuclear gene encoding mitochondrial RNA polymerase. It lacks a mitochondrial targeting signal and localizes to the nucleus, where it is thought to transcribe as many as several hundred genes. Little is known about basal and regulated RNAP-IVSP transcription and its role in nuclear gene expression, yet the existence of a new nuclear RNA polymerase might dramatically change our view of gene regulation.We propose to continue our work on the determination of RNA polymerase specificity at snRNA promoters. We will complete our analysis of how specific recruitment of the TFIIB-related factor Brf2 ensures specific pol III recruitment. To identify additional factors that may favor TFIIB or Brf2 recruitment at pol II and pol III snRNA promoters, respectively, we will develop a method to identify, by mass spectrometry (MS), the proteins present in a chromatin immunoprecipitation (Ch-IP) performed with crosslinked cells (ChIP-MS method). We will use this method to analyze the proteins present on the pol II and III snRNA promoters in vivo as well as proteins involved in pol III and RNAP-IVSP transcription regulation. We will continue our studies on the mammalian pol III repressor Maf1, which like its yeast counterpart is regulated by phosphorylation. We will complete our analysis of the role of Maf1 phosphoresidues in regulating Maf1’s ability to repress pol III. Since we find that human Maf1 is directly phosphorylated by TORC1, and others find TORC1 associated with pol III tRNA genes, we will study the genome-wide localization of both TORC1 and Maf1 with methods we developed for analyzing the mammalian pol III transcriptome. We will continue our characterization of a Maf1-deficient mouse, which is viable and fertile but displays a number of metabolic abnormalities.We will use the RNAP-IVSP in vitro transcription system we developed to define an RNAP-IVSP promoter. We will identify RNAP-IVSP TFs and reveal the genome-wide localization of the enzyme. Together, the proposed experiments will considerably advance our understanding of the regulation of pol III, a key enzyme whose deregulation leads to cancer, and of RNAP-IVSP, a new player in mammalian nuclear gene expression.