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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 162986
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 01.10.2015 - 30.09.2018
Approved amount 834'000.00
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All Disciplines (3)

Discipline
Molecular Biology
Cellular Biology, Cytology
Biochemistry

Keywords (6)

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

Lay Summary (German)

Lead
Die korrekte Expression der genetischen Information ist für die Existenz komplexer Organismen unabdingbar. Um dies zu gewährleisten, haben Zellen eine Vielzahl von Qualitätskontrollmechanismen entwickelt. Die zur Zeit bestuntersuchte dieser Qualitätskontrollen erkennt Boten-RNAs (mRNA) die Probleme bei der Translationstermination haben und wird “nonsense-mediated mRNA decay” (NMD) genannt. Das Projekt hat zum Ziel den molekularen Mechanismus von NMD und dessen physiologische Bedeutung zu verstehen.
Lay summary

Die Umsetzung der genetischen Information, d.h. die Genexpression, basiert eine Kaskade komplexer biochemischer Reaktionen von denen jeder Schritt mit einer gewissen Fehlerrate behaftet ist. Um gesamthaft die Akkumulierung zu vieler Fehler in mRNAs zu vermeiden haben eukaryontische Zellen verschiedene Checkpoints entwickelt, die fehlerhaft prozessierte mRNAs erkennen und rasch degradieren. Einer dieser Checkpoints erkennt mRNAs, deren Proteinbauplan vorzeitig abbricht und für den Prozess des schnellen Abbaus dieser sogenannten nonsense mRNAs wurde der Fachbegriff „nonsense-mediated mRNA decay“ (NMD) geprägt. Über seine Qualitätskontrollfunktion hinaus spielt NMD auch eine Rolle bei der Justierung der intrazellulären Konzentration von ungefähr 10% aller bekannten mRNAs. Welche biologischen Prozessen durch NMD beeinflusst oder gar gesteuert werden, ist noch nicht bekannt und ein Ziel dieses Projekts ist deshalb, mehr über die physiologischen Funktionen von NMD in humanen Zellen zu erfahren. Dazu mutieren und deletieren wir mittels CRISPR-Cas Technologie verschiedene NMD Faktoren in induzierbaren pluripotenten Stammzellen (iPSCs) und untersuchen dann,ob diese Veränderungen einen Einfluss auf die Differenzierung der Stammzellen haben. Ein anderer Fokus liegt auf der Aufklärung des molekularen Mechanismus von NMD. Insbesondere versuchen wir die Kriterien zu verstehen, welche die Zellen zur Unterscheidung zwischen „richtig“ (kein NMD Substrat) und „falsch“ (mRNA wird durch NMD abgebaut) anwendet.

Die erwarteten Resultate sollen nicht nur unser Verständnis für die der Genexpression in menschlichen Zellen unterliegenden molekularen Vorgänge erweitern, sondern sind auch von medizinischem Interesse, da ca. ein Drittel aller mit Krankheiten assoziierten Mutationen zur Produktion von nonsense mRNAs führt, die dann durch NMD abgebaut werden. Aus diesem Grund ist NMD ein wichtiger Modulator der klinischen Manifestationen dieser Erbkrankheiten.
Direct link to Lay Summary Last update: 02.10.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells
Nicholson Pamela, Gkratsou Asimina, Josi Christoph, Colombo Martino, Mühlemann Oliver (2018), Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells, in RNA, 24(4), 557-573.
Beyond quality control: The role of nonsense-mediated mRNA decay (NMD) in regulating gene expression
Nasif Sofia, Contu Lara, Mühlemann Oliver (2018), Beyond quality control: The role of nonsense-mediated mRNA decay (NMD) in regulating gene expression, in Seminars in Cell & Developmental Biology, 75, 78-87.
CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells
Reber Stefan, Mechtersheimer Jonas, Nasif Sofia, Benitez Julio Aguila, Colombo Martino, Domanski Michal, Jutzi Daniel, Hedlund Eva, Ruepp Marc-David (2018), CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells, in Molecular Biology of the Cell, 29(2), 75-83.
New functions in translation termination uncovered for NMD factor UPF3B
Mühlemann Oliver, Karousis Evangelos D (2017), New functions in translation termination uncovered for NMD factor UPF3B, in The EMBO Journal, 36(20), 2928-2930.
Virus Escape and Manipulation of Cellular Nonsense-Mediated mRNA Decay.
Balistreri Giuseppe, Bognanni Claudia, Mühlemann Oliver (2017), Virus Escape and Manipulation of Cellular Nonsense-Mediated mRNA Decay., in Viruses, 9(1), 24.
Minor intron splicing is regulated by FUS and affected by ALS‐associated FUS mutants
Reber Stefan, Stettler Jolanda, Filosa Giuseppe, Colombo Martino, Jutzi Daniel, Lenzken Silvia C, Schweingruber Christoph, Bruggmann Rémy, Bachi Angela, Barabino Silvia ML, Mühlemann Oliver, Ruepp Marc‐David (2016), Minor intron splicing is regulated by FUS and affected by ALS‐associated FUS mutants, in The EMBO Journal, 35(14), 1504-1521.
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), 0150239-0150239.
Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact.
Karousis Evangelos D, Nasif Sofia, Mühlemann Oliver (2016), Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact., in Wiley interdisciplinary reviews. RNA, 7(5), 661-82.
Spermatogenesis Studies Reveal a Distinct Nonsense-Mediated mRNA Decay (NMD) Mechanism for mRNAs with Long 3'UTRs.
Mühlemann Oliver (2016), Spermatogenesis Studies Reveal a Distinct Nonsense-Mediated mRNA Decay (NMD) Mechanism for mRNAs with Long 3'UTRs., in PLoS genetics, 12(5), 1005979-1005979.
Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways.
Colombo Martino, Karousis Evangelos D, Bourquin Joël, Bruggmann Rémy, Mühlemann Oliver (2016), Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways., in RNA (New York, N.Y.), 23(2), 189-201.

Collaboration

Group / person Country
Types of collaboration
Marieke van de Ven, NKI Amsterdam Netherlands (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
Volker Thiel, Vetsuisse, Univ. of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Manfred Heller, PMSCF Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Angela Bachi, IFOM, Milano Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Giuseppe Balistreri, Helsinki University Finland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Fred Allain, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Rémy Bruggmann, IZB, Uni Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Roland Beckmann, LMU Munich Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Jiri Novacek, CEITEC Brno Czech Republic (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Yoon Ki Kim, Korea University, Seoul Korean Republic (South Korea) (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Marc Bühler, FMI Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
CSHL Translation meeting Talk given at a conference Talk by Giuditta Annibaldis: Depletion of the ribosome recycling factor ABCE1 inhibits nonsense-mediated mRNA decay 04.09.2018 Cold Spring Harbor, NY, United States of America Mühlemann Oliver;
FASEB SRC on RNA turnover Talk given at a conference Talk OM: Unraveling the link between translation termination and nonsense-mediated mRNA decay; poster LC: Investigating the role of NMD as an innate cellular antiviral defence mechanism 24.06.2018 Scottsdale, AZ, United States of America Mühlemann Oliver; Contu Lara;
Swiss RNA Workshop 2018 Talk given at a conference OM is a co-organiser of this event, our group presented 7 posters 02.02.2018 Bern, Switzerland Domanski Michal; Nasif Sofia; Bognanni Claudia Ilaria; Contu Lara; Mühlemann Oliver;
RiboClub meeting 2017 Talk given at a conference OM was co-organiser and gave a talk, MD and SN presented posters 24.09.2017 Orford, Canada Mühlemann Oliver; Nasif Sofia; Domanski Michal;
mRNA turnover meeting Talk given at a conference Talk by OM: Comparing translation termination on “normal” and NMD-sensitive mRNAs; poster by LC: A degron-based approach to modulate UPF1 levels in induced pluripotent stem cells (iPSCs) 10.07.2017 Oxford, Great Britain and Northern Ireland Mühlemann Oliver; Contu Lara;
Keystone meeting 2017 on mRNP biology Poster A Toolbox for Purification and Characterization of mRNPs 05.02.2017 Banff, Canada Domanski Michal;
Swiss RNA Workshop 2017 Poster OM is a co-organiser of this event, our group presented six posters 27.01.2017 Bern, Switzerland Domanski Michal; Nasif Sofia; Contu Lara; Mühlemann Oliver;
Cold Spring Harbour Asia Conference “RNA Biology” Talk given at a conference Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways 14.11.2016 Suzhou, China Mühlemann Oliver;
EMBL Symposium: The Complex Life of mRNA Poster Investigation of the role of PNRC2 in nonsense-mediated mRNA decay in human cells 05.10.2016 Heidelberg, Germany Nasif Sofia; Mühlemann Oliver;
Cold Spring Harbor Laboratory: Translational Control Poster In vitro study of aberrant translation termination in the context of a long 3΄UTR 06.09.2016 Cold Spring Harbour, New York, United States of America Mühlemann Oliver;
FASEB SRC on "Post-transcriptional control of gene expression: Mechanisms of RNA decay" Talk given at a conference What makes an NMD target? Identification and characterization of NMD sensitive endogenous transcripts in human cells 10.07.2016 Lisbon, Portugal Bognanni Claudia Ilaria; Mühlemann Oliver;
Swiss RNA Workshop 2016 Poster Production of human cell lines with mutations and deletions in endogenous NMD factors using CRISPR/Cas genome editing 22.01.2016 Bern, Switzerland Mühlemann Oliver; Nasif Sofia;
Swiss RNA Workshop 2016 Poster Investigation of the interaction between UPF1 and the THO/TREX complex 22.01.2016 Bern, Switzerland Mühlemann Oliver;
Swiss RNA Workshop 2016 Poster Molecular study of aberrant translation termination in mammalian systems 22.01.2016 Bern, Switzerland Mühlemann Oliver;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
Kantonale Gymnasialleherertagung Talk 05.12.2017 Bern, Switzerland Mühlemann Oliver;


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Nacht der Forschung German-speaking Switzerland 2017
Media relations: print media, online media Von Molekülen zu Medikamenten UniPress German-speaking Switzerland 2015

Associated projects

Number Title Start Funding scheme
141735 NCCR RNA & disease: Understanding the role of RNA biology in disease mechanisms (phase I) 01.05.2014 National Centres of Competence in Research (NCCRs)
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)
182831 Towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD) 01.10.2018 Project funding (Div. I-III)

Abstract

The term “Nonsense-Mediated mRNA Decay” (NMD) was originally coined to describe 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 serves as a quality control of gene expression and protects the cell from accumulating C-terminally truncated proteins with potentially toxic functions. However, a more general role of NMD in posttranscriptional regulation of gene expression emerged from transcriptome-wide mRNA profilings that identified many physiological (i.e. PTC-free) mRNAs as NMD targets, overall affecting the mRNA levels of 3 - 10% of all genes in yeast, Drosophila, and human cells. 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.Over the past 10 years, our lab has contributed both to the dissection of the molecular mechanism of NMD as well as to a better understanding of the biological function of NMD in mammalian cells. With the projects proposed here, we aim at continuing our research along these two lines by using a combination of biochemical, molecular biology, cell biology and reverse genetics methods. We plan to generate several inducible pluripotent stem cell (iPSC) lines with mutations in various NMD factors and assess their effect on the transcriptome and the capability of the cells to differentiate. The mutations will be introduced using recently developed genome-editing techniques (CRISPR/Cas). Compared to traditional knockdown-rescue experiments, the genome editing approach has the advantage that the effect of the mutant proteins can be assessed in the absence of any remaining low levels of WT protein possibly confounding the phenotype. On the biochemical side, one of our main goals is to establish a protocol to purify specific messenger ribonucleoprotein (mRNP) populations that have been arrested at different stages along the NMD pathway and characterizing their composition by mass spectrometry. This will give us important insight into mRNP remodeling events during NMD. A third line of research concerns a follow-up of our recent finding that NMD appears to play a role in defending cells from RNA virus replication. We want to find out what makes the Semliki Forest Virus genomic RNA an NMD substrate and test other viruses for their sensitivity to NMD.Collectively, our research aims at understanding the molecular mechanism of NMD and its physiological role in human cells. This is the basis for the future development of highly specific approaches to manipulate in a controlled way NMD activity in different disease contexts.
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