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Studies on the role of RNAi and other RNA turnover mechanisms in initiation and maintenance of heterochromatin

English title Studies on the role of RNAi and other RNA turnover mechanisms in initiation and maintenance of heterochromatin
Applicant Bühler Marc
Number 119193
Funding scheme SNSF Professorships
Research institution Friedrich Miescher Institute for Biomedical Research
Institution of higher education Institute Friedrich Miescher - FMI
Main discipline Molecular Biology
Start/End 01.06.2008 - 31.05.2012
Approved amount 1'573'906.00
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All Disciplines (5)

Discipline
Molecular Biology
Cellular Biology, Cytology
Genetics
Molecular Biology
Biochemistry

Keywords (11)

RNA interference; Epigenetics; nuclear RNA turnover; RNA silencing; PEV; chromosome segregation; co-transcriptional gene silencing; chromatin; gene expression; chromosomal architecture; gene expression control

Lay Summary (English)

Lead
Lay summary
Hypothesis: RNA turnover mechanisms contribute to assembly, maintenance, and silencing of heterochromatic domains.Eukaryotic chromosomes contain stretches of a specialized type of chromatin called heterochromatin, or silent chromatin. Heterochromatin is associated with landmark chromosome structures that are required for stable chromosome transmission, and heterochromatin-based gene silencing mechanisms are involved in stable inactivation of genes during development and differentiation. RNA interference (RNAi) is a highly conserved, sequence-specific gene regulatory mechanism among eukaryotes, critical for a variety of important biological functions, including defense against viruses, regulation of gene expression during development, and is being pursued as a promising new tool for the treatment of a variety of human maladies including cancer, neurodegenerative diseases, and viral infections. A surprising link between heterochromatin and the RNAi pathway was discovered a few years ago in fission yeast, and similar mechanisms have more recently been described in various eukaryotes, including plants, Drosophila melanogaster, chicken and Tetrahymena thermophila. Thus, RNAi-mediated chromatin modification leading to heterochromatic gene silencing seems to be a widespread phenomenon in eukaryotes. Biochemical and genetic analyses improved our understanding of how the RNAi pathway contributes to heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and challenged the longstanding paradigm that heterochromatin is an inert, transcriptionally inactive structure. Our recent work has demonstrated that silencing of genes inserted into heterochromatin and heterochromatic repeat elements itself might be mediated by RNAi as well as exosome-mediated co-transcriptional processing events, rather than by shutting off transcription. This novel mode of gene silencing is referred to as “Co-Transcriptional Gene Silencing” (CTGS) and its mechanistic details remain largely unknown. The goal of this project is to completely understand the mechanistic details of CTGS, which will further our understanding of the role of RNA turnover pathways in controlling eukaryotic gene expression. We will also continue analyzing the RNAi machinery by employing parallel genetic, biochemical, advanced live cell imaging and proteomic approaches. We will continue our work by using fission yeast as a model system. We are particularly interested in S. pombe because its heterochromatin has many of the same post-translational modifications and protein components as mammalian systems. In contrast, the RNAi pathway and some of the heterochromatin-specific histone modifications that are shared between S. pombe and metazoans appear to be absent in S. cerevisiae. However, some of the insights gained from working with yeast we will investigate in higher eukaryotes specifically. In summary, we believe that this project will help to elucidate how heterochromatin is assembled, how gene expression within heterochromatin is inactivated and how this is regulated by the RNAi and other RNA processing pathways.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
RNAi keeps Atf1-bound stress response genes in check at nuclear pores.
Woolcock Katrina J, Stunnenberg Rieka, Gaidatzis Dimos, Hotz Hans-Rudolf, Emmerth Stephan, Barraud Pierre, Bühler Marc (2012), RNAi keeps Atf1-bound stress response genes in check at nuclear pores., in Genes & development, 26(7), 683-92.
An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer.
Barraud Pierre, Emmerth Stephan, Shimada Yukiko, Hotz Hans-Rudolf, Allain Frédéric H-T, Bühler Marc (2011), An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer., in The EMBO journal, 30(20), 4223-35.
Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe.
Woolcock Katrina J, Gaidatzis Dimos, Punga Tanel, Bühler Marc (2011), Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe., in Nature structural & molecular biology, 18(1), 94-9.
Proteomic and functional analysis of the noncanonical poly(A) polymerase Cid14.
Keller Claudia, Woolcock Katrina, Hess Daniel, Bühler Marc (2010), Proteomic and functional analysis of the noncanonical poly(A) polymerase Cid14., in RNA (New York, N.Y.), 16(6), 1124-9.
Ribonucleic acid brings chromatin into shape
Bühler Marc (2010), Ribonucleic acid brings chromatin into shape, in The Biochemist.
Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation.
Punga Tanel, Bühler Marc (2010), Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation., in EMBO molecular medicine, 2(4), 120-9.
Nuclear retention of fission yeast dicer is a prerequisite for RNAi-mediated heterochromatin assembly.
Emmerth Stephan, Schober Heiko, Gaidatzis Dimos, Roloff Tim, Jacobeit Kirsten, Bühler Marc (2010), Nuclear retention of fission yeast dicer is a prerequisite for RNAi-mediated heterochromatin assembly., in Developmental cell, 18(1), 102-13.
Silent chromatin at the middle and ends: lessons from yeasts.
Bühler Marc, Gasser Susan M (2009), Silent chromatin at the middle and ends: lessons from yeasts., in The EMBO journal, 28(15), 2149-61.
RNA turnover and chromatin-dependent gene silencing.
Bühler Marc (2009), RNA turnover and chromatin-dependent gene silencing., in Chromosoma, 118(2), 141-51.
HP1Swi6 mediates the recognition and destruction of heterochromatic RNA transcripts
Keller Claudia, Adaixo Ricardo, Stunnenberg Rieka, Woolcock Katrina, Hiller Sebastian, Bühler Marc, HP1Swi6 mediates the recognition and destruction of heterochromatic RNA transcripts, in Molecular Cell.
Schizosaccharomyces pombe reporter strains for relative quantitative assessment of heterochromatin silencing
Shimada Yukiko, Bühler Marc, Schizosaccharomyces pombe reporter strains for relative quantitative assessment of heterochromatin silencing, in Yeast.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
EMBO-Singapore Partnership Launch Symposium 16.03.2012 Singapore
Keystone Symposia “Gene Silencing by Small RNAs” 07.02.2012 Vancouver, Canada
RNA Club 2011, Keynote lecture 11.11.2011 Prague (CZ)
Boehringer Ingelheim Fonds Intl. Titisee Conferences “Genomic Regulation” 2011 12.10.2011 Titisee, Germany
Royal Society “Theo Murphy international scientific meeting on ncRNA” 18.09.2011 London, UK
EMBO Young Scientists Forum, 2011 30.06.2011 Warsaw, Poland
Keystone Symposia “Mechanism and Biology of Silencing”, 2011 20.03.2011 Monterey, USA
5th microsymposium on small RNAs, 2010 17.05.2010 Vienna, Austria
4th international workshop on cell regulations in division and arrest, 2009 29.11.2009 Okinawa Japan


Self-organised

Title Date Place
7th microsymposium on small RNAs, 2012 21.05.2012 Basel, CH
FMI 40th Anniversary Symposium 20.09.2010 Basel, CH

Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) Combating fungal diseases German-speaking Switzerland Italian-speaking Switzerland Western Switzerland International Rhaeto-Romanic Switzerland
New media (web, blogs, podcasts, news feeds etc.) Dicer shuttles to nucleus and accumulates to form heterochromatin Rhaeto-Romanic Switzerland Western Switzerland International Italian-speaking Switzerland German-speaking Switzerland
New media (web, blogs, podcasts, news feeds etc.) Inhibiting RNA in the nucleus to stay tuned for an emergency response Web German-speaking Switzerland Italian-speaking Switzerland Rhaeto-Romanic Switzerland Western Switzerland International
Media relations: radio, television Kann Pflanzenerbgut unsere Gene steuern? DRS2 German-speaking Switzerland
New media (web, blogs, podcasts, news feeds etc.) Mechanism elucidated for a rare disease Western Switzerland Rhaeto-Romanic Switzerland International German-speaking Switzerland Italian-speaking Switzerland
Media relations: radio, television Telebar TeleBasel German-speaking Switzerland

Awards

Title Year
ERC starting grant 2011
Friedrich Miescher Prize 2011
EMBO young investigator 2010
Biorad RNAi Award 2008

Associated projects

Number Title Start Funding scheme
139204 Studies on the role of RNAi and other RNA turnover mechanisms in initiation and maintenance of heterochromatin 01.06.2012 SNSF Professorships

Abstract

Hypothesis: RNA turnover mechanisms contribute to assembly, maintenance, and silencing of heterochromatic domains.Eukaryotic chromosomes contain stretches of a specialized type of chromatin called heterochromatin, or silent chromatin. Heterochromatin is associated with landmark chromosome structures that are required for stable chromosome transmission, and heterochromatin-based gene silencing mechanisms are involved in stable inactivation of genes during development and differentiation. RNA interference (RNAi) is a highly conserved, sequence-specific gene regulatory mechanism among eukaryotes, critical for a variety of important biological functions, including defense against viruses, regulation of gene expression during development, and is being pursued as a promising new tool for the treatment of a variety of human maladies including cancer, neurodegenerative diseases, and viral infections. A surprising link between heterochromatin and the RNAi pathway was discovered a few years ago in fission yeast, and similar mechanisms have more recently been described in various eukaryotes, including plants, Drosophila melanogaster, chicken and Tetrahymena thermophila. Thus, RNAi-mediated chromatin modification leading to heterochromatic gene silencing seems to be a widespread phenomenon in eukaryotes. Biochemical and genetic analyses improved our understanding of how the RNAi pathway contributes to heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and challenged the longstanding paradigm that heterochromatin is an inert, transcriptionally inactive structure. My recent work has demonstrated that silencing of genes inserted into heterochromatin and heterochromatic repeat elements itself might be mediated by RNAi as well as exosome-mediated co-transcriptional processing events, rather than by shutting off transcription. This novel mode of gene silencing is referred to as “Co-Transcriptional Gene Silencing” (CTGS) and its mechanistic details remain largely unknown. My goal is to completely understand the mechanistic details of CTGS, which will further our understanding of the role of RNA turnover pathways in controlling eukaryotic gene expression. I will also continue analyzing the RNAi machinery by employing parallel genetic, biochemical, molecular biological, and proteomic approaches. My detailed research plan is divided into four specific aims: A. Mechanistic dissection of CTGS. In fission yeast, RNAi- and exosome-dependent RNA turnover mechanisms are required to keep heterochromatic domains silent. My current work shows that the polyA polymerase Cid14 mediates the degradation of heterochromatic transcripts by these pathways. However, the exact mechanism remains unknown. Important questions remain: how are heterochromatic transcripts recognized by Cid14? Are heterochromatic transcripts marked by Cid14 with a specific polyA signature? What specifies whether RNAi or the exosome degrades the Cid14 substrate? In which compartment (nucleus versus cytoplasm) does RNA degradation occur? What is the role of polyadenylation in the RNAi pathway? These questions will be addressed by employing parallel genetic, biochemical, molecular biological, and proteomic approaches and will advance our mechanistic understanding of heterochromatic gene silencing.B. Comprehensive analysis of Cid14-mediated gene silencing. The polyadenylation activity of Cid14 has also been implicated in ribosomal RNA processing and it is likely to be part of other RNA processing mechanisms. In order to get a more comprehensive picture of Cid14 function, a genomics approach will be employed to analyze Cid14-mediated gene silencing in fission yeast on a genome-wide scale. This aim will address the question whether Cid14-mediated CTGS is a general mechanism to keep heterochromatin “silent” and is likely to uncover further examples of gene regulation by polyadenylation assisted RNA turnover. C. Visualizing heterochromatic transcripts in live yeast cells. Genetic and biochemical experiments have contributed substantially to our understanding of heterochromatic gene silencing mechanisms. The finding that heterochromatin can be transcribed revealed new questions which are difficult to address by genetics and biochemistry alone. Therefore, I am eager to use microscopy to address problems such as the cellular localization of heterochromatic transcript degradation, the dynamics of transcription within heterochromatin and changes in the fate of heterochromatic structures as well as transcripts throughout the cell cycle. The focus of this aim is to develop a technology that is essential for addressing the most intriguing questions in the field that have so far been unanswerable. Furthermore, by doing so, I will further differentiate myself from other labs working on the same topic. D. Identification of negative regulators of RNAi-mediated heterochromatin assembly. One of the key features of RNAi-mediated heterochromatic gene silencing in fission yeast is its cis restriction. My work suggests the existence of specific factors that restrict RNAi-mediated heterochromatin formation in cis. In order to identify novel negative regulators of RNAi-mediated heterochromatin assembly, I will perform genetic screens using a system I developed as a post-doctoral researcher. This aim could potentially lead to the identification of novel factors regulating the RNAi pathway and its link to chromatin. In addition, this aim has also a great potential for new projects to emerge.I am fascinated by the recent discoveries that put RNA into a new light. The discovery of the RNAi pathway is, in my opinion, one of the most exciting events in the history of gene regulation. RNAi brings together nearly every aspect of gene regulation and is likely to have far-reaching implications for our understanding of how genomes evolved and are regulated. I am particularly interested in understanding the link between RNA turnover mechanisms and epigenetics. In contrast to the silencing response that operates at a posttranscriptional level, we are only at the beginning of genetic studies leading the way toward a model in which closely related complexes are also involved in silencing at the genomic level and its biochemical analysis is a big challenge for me. I will continue my work by using fission yeast as a model system. I am particularly interested in S. pombe because its heterochromatin has many of the same post-translational modifications and protein components as mammalian systems. However, I wish to start investigating some of the insights gained from working with yeast in higher eukaryotes as well.
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