Project

Back to overview

Quality control of gene expression: towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD)

English title Quality control of gene expression: towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD)
Applicant Mühlemann Oliver
Number 143717
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 01.10.2012 - 30.09.2015
Approved amount 630'000.00
Show all

All Disciplines (3)

Discipline
Molecular Biology
Biochemistry
Cellular Biology, Cytology

Keywords (6)

RNA quality control; mRNA turnover; posttranscriptional gene regulation; Nonsense-mediated mRNA decay; mRNA surveillance; translation termination

Lay Summary (English)

Lead
Lay summary

Accurate expression of the genetic information is crucial for complex organisms and requires several different quality control (QC) mechanisms that recognize mistakes along the cascade of complex biochemical reactions from RNA synthesis to functional proteins. Among those, nonsense-mediated mRNA decay (NMD) has been initially characterized as a translation-dependent posttranscriptional QC system that selectively recognizes and degrades mRNAs with crippled protein-coding potential, thereby preventing the cell from production of truncated proteins with potentially toxic functions. Recent transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that NMD affects the mRNA concentrations of 3-10% of all genes, indicating a role of NMD in gene regulation that extends beyond QC. NMD is essential in vertebrates and an important modulator of genetic disease phenotypes in humans, since about 30% of all known disease-causing mutations trigger NMD. While the phenomenon of NMD and its impact on gene expression and genetic diseases is well documented, the understanding of the underlying molecular mechanisms is still fragmented

The specific projects supported by this grant are a continuation of more than 10 years of SNF-supported basic research aiming at unraveling the molecular mechanism and physiological role of NMD in human cells by using a combination of state-of-the-art biochemical, molecular biology, cell biology and reverse genetics methods. Specifically, we try to understand the features that identify an mRNA as a substrate for NMD and want to know which factors interact how, where and in which temporal order with the these mRNAs and with each other to achieve the rapid degradation of the target mRNAs. In this context, we also try to decipher the puzzling nuclear effects observed with NMD-targeted mRNAs. Given its tight coupling to translation, NMD is commonly assumed to occur in the cytoplasm, and the recurring effects of NMD-causing premature termination codons on nuclear processes like transcription and splicing are therefore enigmatic. Furthermore, we attempt to reveal the roles that NMD plays in regulating gene expression. To this end, we want to identify and characterize transcriptome-wide mRNAs differentially regulated by NMD during the differentiation of neuroblastoma into neuron-like cells and thereby discover specific biological pathways that are controlled by NMD. In particular, we will to test the hypothesis that spatial rearrangements of the 3’ UTR configuration represent a way by which the half-life of mRNAs can be differentially regulated by NMD.

Concerning planned methodological progress, we will expand and optimize the use of lentiviral constructs to engineer cell lines that allow inducible expression of two or even three transgenes separately. We need such cell lines for the planned purification and characterization of specific mRNP populations that have been arrested at different stages along the NMD pathway. If successful, this method will have a wide range of interesting applications beyond NMD research.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID)
Schweingruber Christoph, Soffientini Paolo, Ruepp Marc-David, Bachi Angela, Mühlemann Oliver (2016), Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID), in PLoS one, 11(3), e0150239.
A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD.
Nicholson Pamela, Josi Christoph, Kurosawa Hitomi, Yamashita Akio, Mühlemann Oliver (2014), A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD., in Nucleic acids research, 42(14), 9217-35.
Characterization of phosphorylation- and RNA-dependent UPF1 interactors by quantitative proteomics.
Flury Valentin, Restuccia Umberto, Bachi Angela, Mühlemann Oliver (2014), Characterization of phosphorylation- and RNA-dependent UPF1 interactors by quantitative proteomics., in Journal of proteome research, 13(6), 3038-53.
Eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay by two genetically separable mechanisms.
Joncourt Raphael, Eberle Andrea B, Rufener Simone C, Mühlemann Oliver (2014), Eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay by two genetically separable mechanisms., in PloS one, 9(8), 104391-104391.
Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1
David Zünd, Oliver Mühlemann (2014), Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1, Bioprotocols, Sunnyvale, CA, USA.
The host nonsense-mediated mRNA decay pathway restricts Mammalian RNA virus replication.
Balistreri Giuseppe, Horvath Peter, Schweingruber Christoph, Zünd David, McInerney Gerald, Merits Andres, Mühlemann Oliver, Azzalin Claus, Helenius Ari (2014), The host nonsense-mediated mRNA decay pathway restricts Mammalian RNA virus replication., in Cell host & microbe, 16(3), 403-11.
UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells
David Zünd, Oliver Mühlemann (2014), UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells, Bioprotocols, Sunnyvale, CA, USA.
Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways.
Metze Stefanie, Herzog Veronika A, Ruepp Marc-David, Mühlemann Oliver (2013), Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways., in RNA (New York, N.Y.), 19(10), 1432-48.
eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells.
Rufener Simone C, Mühlemann Oliver (2013), eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells., in Nature structural & molecular biology, 20(6), 710-7.
Nonsense-mediated mRNA decay - mechanisms of substrate mRNA recognition and degradation in mammalian cells.
Schweingruber Christoph, Rufener Simone C, Zünd David, Yamashita Akio, Mühlemann Oliver (2013), Nonsense-mediated mRNA decay - mechanisms of substrate mRNA recognition and degradation in mammalian cells., in Biochimica et biophysica acta, 1829(6-7), 612-23.
Recent transcriptome-wide mapping of UPF1 binding sites reveals evidence for its recruitment to mRNA before translation
Zünd David, Mühlemann Oliver (2013), Recent transcriptome-wide mapping of UPF1 binding sites reveals evidence for its recruitment to mRNA before translation, in Translation, 1, e26977.
RNA decay mechanisms: Specifcity through diversity (Editorial for special issue on RNA decay mechanisms).
Georg Stoecklin, Oliver Mühlemann (2013), RNA decay mechanisms: Specifcity through diversity (Editorial for special issue on RNA decay mechanisms)., in Biochem Biophys Acta - Gene Regulatory Mechanisms, 1829(6-7), 487-490.
Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs.
Zünd David, Gruber Andreas R, Zavolan Mihaela, Mühlemann Oliver (2013), Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs., in Nature structural & molecular biology, 20(8), 936-43.
News and Views: Intimate liaison with SR proteins brings exon junction complexes to unexpected places
Oliver Mühlemann (2012), News and Views: Intimate liaison with SR proteins brings exon junction complexes to unexpected places, in Nat Struct Mol Biol, 19(12), 1209-1211.

Collaboration

Group / person Country
Types of collaboration
Akio Yamashita, Yokohama City University Japan (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Silvia Barabino, Univ. Milano-Biccoca Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Stefania Corti, San Raffaele Milano Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Volker Thiel, Vetsuisse, Uni Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Martin Lochner, DCB Uni Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Angela Bachi, San Raffaele, Milano Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Torben Heick Jensen, Århus University Denmark (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Rémy Bruggmann, IZB, Uni Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Ari Helenius & Guiseppe Balistreri, ETH Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
EMBO conference on Protein Synthesis and Translational Control Poster "Evidence for a coupling between mRNA export and translation initiation through CTIF binding to DDX19B" AND "Mechanistic study of premature termination codon recognition in mammalian systems" 09.09.2015 Heidelberg, Germany Karousis Evangelos; Rufener Simone Carla; Mühlemann Oliver;
RNA Stability 2015 Meeting Talk given at a conference Nonsense-mediated mRNA decay (NMD) restricts replication of mammalian RNA viruses 01.06.2015 Estes Park, Co, United States of America Mühlemann Oliver;
RNA 2015 - Annual meeting of the international RNA society Poster NMD restricts replication of mammalian RNA viruses 26.05.2015 Madison, Wi, United States of America Mühlemann Oliver;
IGMM Seminar, CNRS Montpellier Individual talk Trying to make sense in nonsense-mediated mRNA decay 14.10.2014 Montpellier, France Mühlemann Oliver;
FASEB SRC: Post-transcriptional control of gene expression Talk given at a conference Nonsense-mediated mRNA decay (NMD) restricts replication of + strand RNA viruses 06.07.2014 Big Sky, Mo, United States of America Mühlemann Oliver;
RNA 2014 - Annual meeting of the international RNA society Poster CTIF is involved in mRNA export in human cells AND SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1 AND Probing interactions of NMD factors in a distance-dependent manner by BioID 03.06.2014 Quebec, Canada Mühlemann Oliver; Nicholson Pamela; Rufener Simone Carla;
EMBO Conference on Protein Synthesis and Translational Control Talk given at a conference Mechanistic aspects of NMD in human cells 08.09.2013 Heidelberg, Germany Mühlemann Oliver;
RNA 2013 - Annual meeting of the international RNA society Talk given at a conference "eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells" plus 7 presented posters from my group 11.06.2013 Davos, Switzerland Mühlemann Oliver; Rufener Simone Carla; Nicholson Pamela;
EMBO Eucaryotic RNA turnover conference Talk given at a conference Mechanistic aspects of mRNA targeting for nonsense-mediated mRNA decay in human cells 21.04.2013 Strasbourg, France Mühlemann Oliver;
ENS seminar Individual talk Trying to make sense about all this nonsense (-mediated mRNA decay) 02.12.2012 Paris, France Mühlemann Oliver;


Self-organised

Title Date Place
Swiss RNA Workshop 2015 23.01.2015 Bern, Switzerland
Swiss RNA workshop 2014 24.01.2014 Bern, Switzerland
Swiss RNA Workshop 2013 25.01.2013 Bern, Switzerland

Communication with the public

Communication Title Media Place Year
Media relations: print media, online media Am Ursprung des Lebens Berner Zeitung German-speaking Switzerland 2014
Media relations: print media, online media Wie LUCA, die Urzelle des Lebens, entstand VSAO Journal German-speaking Switzerland 2014
Other activities Zukunftstag 2012 und 2013: eintägiges Programm für Schüler German-speaking Switzerland 2012

Associated projects

Number Title Start Funding scheme
162986 Quality control of gene expression: towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD) 01.10.2015 Project funding (Div. I-III)
157704 Direct electron detector and phase plate for cryo-transmission electron microscopy of biological samples 01.12.2014 R'EQUIP
127614 Quality control of gene expression: recognition and elimination of nonsense mRNA 01.10.2009 Project funding (Div. I-III)

Abstract

Accurate expression of the genetic information is achieved with the help of several quality control (QC) systems that recognize mistakes along the cascade of complex biochemical reactions from RNA synthesis to functional proteins and so prevent production of faulty gene products. One of these QC systems is “nonsense-mediated mRNA decay” (NMD), a translation-dependent process that degrades mRNAs with truncated open reading frames (ORFs). By recognizing and degrading mRNAs with premature termination codons (PTCs), many of which arise by alternative splicing, NMD protects the cell from accumulating C-terminally truncated proteins with potentially toxic functions. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that NMD affects (directly or indirectly) the mRNA levels of 3 - 10% of all genes, indicating a role of NMD in gene regulation that extends beyond QC. NMD is essential in vertebrates and an important modulator of genetic disease phenotypes in humans, since 30% of all known disease-causing mutations are predicted to trigger NMD. While the phenomenon of NMD and its impact on gene expression and genetic diseases is well documented, the understanding of the underlying molecular mechanisms is still fragmented.Altogether, the projects proposed here aim at unraveling the molecular mechanism and physiological role of NMD in human cells. We use a combination of state-of-the-art biochemical, molecular biology, cell biology and reverse genetics methods to elucidate which features render an mRNA a NMD substrate, and which factors interact how, where and in which temporal order with these features and with each other to achieve the rapid degradation of target mRNAs. In this context, we also try to decipher the puzzling nuclear effects observed with NMD-targeted mRNAs. Given its tight coupling to translation, NMD is commonly assumed to occur in the cytoplasm, and the recurring NMD factor-dependent effects of PTCs on nuclear processes like transcription and splicing are enigmatic. Besides trying to get further insight into the mechanism of NMD, we also attempt to reveal the roles that NMD plays in regulating gene expression. To this end, we want to identify and characterize transcriptome-wide mRNAs regulated by NMD and thereby discover specific biological functions that are controlled by NMD. In particular, we also want to test the hypothesis that spatial rearrangements of the 3’ UTR configuration represent a way by which the half-life of mRNAs can be differentially regulated by NMD.In terms of methodological progress, we began to utilize lentiviral constructs to engineer cell lines that allow inducible expression of two or even three transgenes separately. We will need such cell lines to achieve our ambitious goal of purifying from cells and characterizing by mass spectrometry the composition of specific mRNP populations that have been arrested a different stages along the NMD pathway. If successful, this method will have a wide range of interesting applications in different areas of the molecular life sciences.
-