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Quality control of gene expression: recognition and elimination of nonsense mRNA

English title Quality control of gene expression: recognition and elimination of nonsense mRNA
Applicant Mühlemann Oliver
Number 127614
Funding scheme Project funding (Div. I-III)
Research institution Departement für Chemie und Biochemie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Molecular Biology
Start/End 01.10.2009 - 30.09.2012
Approved amount 468'000.00
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All Disciplines (3)

Discipline
Molecular Biology
Biochemistry
Cellular Biology, Cytology

Keywords (10)

Nonsense-mediated mRNA decay; mRNA turnover; posttranscriptional gene regulation; translation termination; RNA quality control; mRNA surveillance; Nonsense-mediated mRNA decay (NMD); post-transcriptional gene regulation; RNA turnover; mRNA quality control

Lay Summary (English)

Lead
Lay summary
Different quality control mechanisms exist to recognize mistakes during the molecular processes involved in gene expression and to prevent production of faulty gene products. Among those, nonsense-mediated mRNA decay (NMD) represents a translation-dependent posttranscriptional process that selectively recognizes and degrades mRNAs whose open reading frame (ORF) is truncated by a premature translation-termination codon (PTC, also called "nonsense codon"). In doing so, NMD protects the cell from accumulating C-terminally truncated proteins with potentially deleterious functions. Because 30% of all known disease-causing mutations are predicted to trigger NMD, NMD represents an important modulator of genetic disease phenotypes in humans, and understanding the molecular mechanisms of NMD therefore is key for developing future therapies for many genetic diseases. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that 3-10% of all mRNA levels are affected by NMD, indicating an important role of NMD in gene regulation that extends beyond quality control. While the phenomenon of NMD and its impact on gene expression is well documented, the understanding of the underlying molecular mechanisms is still fragmented.Our research attempts to solve two key questions about NMD:1.) Which features distinguish faulty mRNAs that will be subjected to NMD from "normal" mRNAs that will not be targeted by NMD? How does this substrate recognition work at the molecular level? Specifically, we will test a working model that we recently postulated based on our previous work, the so-called unified NMD model (see Mühlemann et al., Biochim Biophys Acta, 2008). This model posits that proper translation termination depends on stimulating signals from the 3' UTR and that in the absence of these signals, translation termination is slow, giving time for the assembly of a protein complex that mediates NMD. The model further predicts that spatial rearrangements of the 3' UTR structure of an mRNA may represent a novel mechanism for tissue-specific or developmentally regulated post-transcriptional control of gene expression.2.) Once identified as nonsense transcript, how are these mRNAs rapidly eliminated? We have recently discovered that one of the known NMD factors, SMG6, is an endonuclease that can cleave an mRNA internally at the site where it binds the RNA (Eberle et al., Nat Struct Mol Biol, 2009). On the other hand, other laboratories have reported that nonsense mRNAs are degraded exonucleolytically from both ends in human cells. We will scrutinze both decay pathways and and try to determine their relative contribution to NMD.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Analysis of nonsense-mediated mRNA decay in mammalian cells.
Nicholson Pamela, Joncourt Raphael, Mühlemann Oliver (2012), Analysis of nonsense-mediated mRNA decay in mammalian cells., in Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.], Chapter 27, 27-4.
mRNP quality control goes regulatory.
Mühlemann Oliver, Jensen Torben Heick (2012), mRNP quality control goes regulatory., in Trends in genetics : TIG, 28(2), 70-7.
Cutting the nonsense: the degradation of PTC containing mRNAs
Nicholson Pamela, Mühlemann Oliver (2010), Cutting the nonsense: the degradation of PTC containing mRNAs, in Biochem Soc Trans, 38(6), 1615-1620.
tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans
Aeby Eric, Ullu Elisabetta, Yepiskoposyan Hasmik, Schimanski Bernd, Roditi Isabel, Mühlemann Oliver, Schneider André (2010), tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans, in Nucleic Acids Res, 17, 5833-5843.
How and where are nonsense mRNAs degraded in mammalian cells?
Mühlemann Oliver, Lykke-Andersen Jens (2010), How and where are nonsense mRNAs degraded in mammalian cells?, in RNA Biology, 7(1), 28-32.
Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors
Nicholson Pamela, Yepiskoposyan Hasmik, Metze Stefanie, Zamudio Orozco Rodolfo, Kleinschmidt Nicole, Mühlemann Oliver (2009), Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors, in Cell Mol Life Sci, 67(5), 677-700.
Autoregulation of the nonsense-mediated mRNA decay pathway in human cells
Yepiskoposyan Hasmik, Aeschimann Florian, Nilsson Daniel, Okoniewski Michal, Mühlemann Oliver, Autoregulation of the nonsense-mediated mRNA decay pathway in human cells, in RNA.
Co-transcriptional Effects of a Premature Termination Codon Revealed by Live-Cell Imaging
de Turris Valeria, Nicholson Pamela, Zamudio Orozco Rodolfo, Singer Robert H., Mühlemann Oliver, Co-transcriptional Effects of a Premature Termination Codon Revealed by Live-Cell Imaging, in RNA.

Collaboration

Group / person Country
Types of collaboration
Hämatologie Abteilung, Inselspital Bern Switzerland (Europe)
- 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
FASEB SRC on Post-Transcriptional Control of Gene Expression 24.06.2012 Steamboat Springs, CO, USA
17th annual meeting of the international RNA society 29.05.2012 Ann Arbor, MI, USA
TERM 2012 22.03.2012 Gif-sur-Yvette, France
EMBL conference on Translational regulation 11.09.2011 Heidelberg, Germany
EMBO young scientists forum 30.06.2011 Warsaw, Poland
16th annual meeting of the international RNA society 13.06.2011 Kyoto, Japan
TERM 2011 26.05.2011 Gif-sur-Yvette, France
Gordon Conference: The Biology of Post-Transcriptional Gene Regulation 18.07.2010 Newport, USA
FASEB SRC on RNA turnover 11.07.2010 Carefree, Arizona, USA
Post-transcriptional control: mRNA translation, localization and turnover 28.06.2010 Edinburgh, UK
15th annual meeting of the RNA society 22.06.2010 Seattle, USA
ESF – RNAQuality conference 06.05.2010 Vienna, Austria
5th meeting on RNA research in Portugal 05.11.2009 Oeiras, Portugal


Self-organised

Title Date Place
Swiss RNA Workshop 2012 03.02.2012 Bern
Swiss RNA workshop 2011 28.01.2011 Bern
Swiss RNA Workshop 2010 22.01.2010 Bern

Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Seniorenuniversität: Entstehung des Lebens German-speaking Switzerland 27.04.2012
Talks/events/exhibitions DCB Tag der offenen Tür German-speaking Switzerland 18.06.2011
Talks/events/exhibitions Nacht der Forschung: Leben ist Chemie German-speaking Switzerland 23.09.2011
Talks/events/exhibitions BioChemie am Samastag: Entstehung des Lebens German-speaking Switzerland 20.11.2010
Talks/events/exhibitions Entstehung des Lebens German-speaking Switzerland 24.05.2010

Associated projects

Number Title Start Funding scheme
139231 Advancement of functional genomics research at the University of Bern by extension of LC-MS platform 01.07.2012 R'EQUIP
113878 Quality control of gene expression: mechanisms for recognition and elimination of nonsense mRNA 01.10.2006 Project funding (Div. I-III)
133813 Next generation sequencing platform, University of Bern 01.12.2010 R'EQUIP
143717 Quality control of gene expression: towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD) 01.10.2012 Project funding (Div. I-III)

Abstract

Background:Many different quality control mechanisms have evolved to recognize mistakes during the molecular processes involved in gene expression and to prevent production of faulty gene products. Among those, nonsense-mediated mRNA decay (NMD) represents a translation-dependent posttranscriptional process that selectively recognizes and degrades mRNAs whose open reading frame (ORF) is truncated by a premature translation-termination codon (PTC, also called „nonsense codon“). In doing so, NMD protects the cell from accumulating C-terminally truncated proteins with potentially deleterious functions. Because 30% of all known disease-causing mutations are predicted to trigger NMD, NMD represents an important modulator of genetic disease phenotypes in humans, and understanding the molecular mechanisms of NMD therefore is key for developing future therapies for many genetic diseases. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that 3-10% of all mRNA levels are regulated (directly or indirectly) by NMD, indicating an important role of NMD in gene regulation that extends beyond quality control. While the phenomenon of NMD and its impact on gene expression is well documented, the understanding of the underlying molecular mechanisms is still fragmented.Current and future research on nonsense-mediated mRNA decay (NMD):A central question of NMD research is how PTCs are recognized and discriminated from natural termination codons. Recent work from my lab and others led to a new model postulating an evolutionarily conserved mechanism for PTC recognition. This so-called “unified NMD model” and its implications for posttranscriptional gene regulation and certain genetic diseases need to be critically scrutinized in the coming years. To further investigate the molecular mechanism of PTC recognition, we concentrate on trying to understand the PABP-mediated promotion of translation termination and the exact role of UPF1 in NMD. For UPF1, we plan to systematically identify interaction partners during the different stages of NMD complex assembly with help of a new mass spectrometry-based technique that enables quantitative identification of interacting factors from affinity purified complexes (SILAC-IP). Another goal is to identify endogenous examples of the postulated posttranscriptional mechanism for gene regulation by spatial rearrangement of the 3’ UTR, a direct implication of the “unified NMD model”. Along these lines, we will also try to identify disease-related human mutations that change the 3’ UTR of the mRNA and experimentally validate those transcripts predicted by our model to have become NMD substrates. Substantial progress in understanding the degradation of nonsense mRNAs in human cells has been made by our recent demonstration that SMG6 is an endonuclease and cleaves the transcript near the PTC. In the future, we want to address if decay of all human NMD substrates is induced by SMG6-mediated endonucleolytic cleavage, or if additional parallel pathways exist. In the case of multiple pathways, we would like to identify the decisive parameters for one or another pathway.Nonsense-mediated transcriptional gene silencing (NMTGS) - quo vadis?Transcriptional silencing of PTC-containing Ig-mu minigenes stably integrated into the genome of HeLa cells was serendipitously discovered in our lab five years ago. Although the biological relevance of this observation still remains elusive, the mechanism itself is intriguing: a translation signal on an mRNA (the PTC) somehow feeds back to its cognate gene and silences its transcription. We are currently trying to establish a more robust experimental system that would enable us to continue our mechanistic dissection of NMTGS. Circumstantial evidence suggests the involvement of small RNAs and components of the RNAi machinery, but more direct evidence is needed. Various experiments are planned to gain more insight into this enigmatic process.
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