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Mechanisms of basal and regulated RNA polymerase III transcription in mammalian cells

English title Mechanisms of basal and regulated RNA polymerase III transcription in mammalian cells
Applicant Hernandez Nouria
Number 132958
Funding scheme Project funding (Div. I-III)
Research institution Centre Intégratif de Génomique Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Molecular Biology
Start/End 01.11.2010 - 31.10.2013
Approved amount 755'000.00
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All Disciplines (2)

Discipline
Molecular Biology
Biochemistry

Keywords (7)

RNA polymerase III; Maf1; Brf2; transcription regulation; U6 snRNA gene; TORC1; genome

Lay Summary (English)

Lead
Lay summary
The first step to decipher an instruction encoded in the genome is the process of transcription, in which a gene is read and "transcribed" into an RNA molecule. This step is highly regulated because often, the decision to transcribe a gene leads to the execution of the instruction encoded in the gene. The basal transcription machinery is complex and still only partially understood. It is controlled by a large number of molecules that receive signals, either from outside or from inside the cell, and then transmit them to either activate or repress it. This project is concerned with deciphering mechanisms of basal and regulated transcription. The core of the basal transcription machinery is the RNA polymerase (pol), a large enzyme capable of polymerizing an RNA chain based on a DNA template. Of the three main nuclear pols, this project focuses mostly on pol III, which transcribes an eclectic collection of short genes encoding RNAs required for basic cell metabolism. These genes have characteristic promoter sequences that indicate where and when transcription begins. Pol III, like pol I and II, is incapable of recognizing its target promoters on its own. Rather, it relies on the help of a set of accessory transcription factors. In this granting period, we focus on four broad questions. The first concerns the determination of pol specificity: why does a certain promoter sequence recruit pol III rather than, in our case, pol II? The results will further our understanding of how promoter sequences direct the formation of specific assemblies of transcription factors which, in turn, are capable of recruiting a specific polymerase. The second point of focus concerns the regulation of pol III transcription by a specific repressor protein called Maf1. Pol III activity is regulated with cell growth and proliferation, but the mechanisms enforcing this regulation are only partially understood. We want to decipher, at the molecular level, how Maf1 participates in this regulation. The third question concerns the role of Maf1 in the context of an entire organism. To study this question, we have obtained mice that lack the gene coding for Maf1 and are studying study them to define any phenotype related to the lack of the Maf1 gene. These studies will reveal mechanisms of pol III transcription regulation that are, ultimately, necessary for controlled cell growth and cell division, and that are deregulated in cancers. The fourth subject of our study is the role that a recently described RNA polymerase, called RNAP-IVsp, might play in the human cell, and which mechanisms regulate its activity.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Gene duplication and neofunctionalization: POLR3G and POLR3GL.
Renaud Marianne, Praz Viviane, Vieu Erwann, Florens Laurence, Washburn Michael P, L'hôte Philippe, Hernandez Nouria (2014), Gene duplication and neofunctionalization: POLR3G and POLR3GL., in Genome research, 24, 37-51.
Eeny meeny miny moe, Catch a transcript by the toe, or How to Enumerate Eukaryotic Transcripts
Strick Térence R., Hernandez Nouria (2012), Eeny meeny miny moe, Catch a transcript by the toe, or How to Enumerate Eukaryotic Transcripts, in Genes and Development, 26(15), 1643-1647.
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.
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, 8(11), 1003028-1003028.
Nanopore detection of single molecule RNAP-DNA transcription complex.
Raillon C, Cousin P, Traversi F, Garcia-Cordero E, Hernandez N, Radenovic A (2012), Nanopore detection of single molecule RNAP-DNA transcription complex., in Nano letters, 12(3), 1157-64.
MAF1: a new target of mTORC1
Michels Annemieke (2011), MAF1: a new target of mTORC1, in Biochem. Soc. Trans., 39(2), 487-491.
Widespread occurrence of non-canonical transcription termination by human RNA polymerase III
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, 39(13), 5499-5512.

Collaboration

Group / person Country
Types of collaboration
Swiss Institute of Bioinformatics Switzerland (Europe)
- Publication
Università degli Studi di Parma Italy (Europe)
- Publication
University of Basel Biozentrum Switzerland (Europe)
- Publication
Albert Einstein College of Medicine United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
8th International Biennial Conference on RNA Polymerases I and III Individual talk A Multiplicity of Factors Contributes to Selective RNA polymerase III occupancy of a Subset of RNA polymerase III Genes in Mouse Liver 07.07.2012 Warrenton, VA, USa, United States of America Hernandez Nouria;
conference on Gene Transcription in Yeast Individual talk A Multiplicity of Factors Contributes to Selective RNA polymerase III occupancy of a Subset of RNA polymerase III Genes in Mouse Liver 16.06.2012 St. Feliu de Guixols, Spain, Spain Hernandez Nouria;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
women in science luncheon Talk 11.10.2012 Montreux, Switzerland Hernandez Nouria;


Associated projects

Number Title Start Funding scheme
145002 Microscale thermophoresis for the Faculty of Bology and Medicine in Lausanne 01.12.2012 R'EQUIP
149904 Mechanisms of basal and regulated mammalian RNA polymerase III transcription 01.11.2013 Project funding (Div. I-III)
125230 Revealing RNA polymerase II and III transcriptional programs in human cells 01.08.2009 Sinergia
150827 High resolution, high sensitivity tandem mass spectrometry for proteomics in a core facility 01.01.2014 R'EQUIP
109941 Mechanisms of basal and regulated RNA polymerase III transcription in mammalian cells 01.11.2005 Project funding (Div. I-III)

Abstract

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.
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