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Quantifying gene expression noise via single mRNA imaging in living cells

Applicant Voigt Franka
Number 171377
Funding scheme Marie Heim-Voegtlin grants
Research institution Friedrich Miescher Institute for Biomedical Research
Institution of higher education Institute Friedrich Miescher - FMI
Main discipline Cellular Biology, Cytology
Start/End 01.02.2017 - 31.05.2019
Approved amount 225'003.00
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All Disciplines (4)

Discipline
Cellular Biology, Cytology
Biochemistry
Molecular Biology
Biophysics

Keywords (5)

Gene expression noise; Translation; mRNA degradation; Posttranscriptional gene expression regulation; Single-molecule mRNA imaging

Lay Summary (German)

Lead
Genexpression bezeichnet das Ablesen und Umsetzen der genetischen Information im Organismus. Es ist ein hochregulierter Prozess, der mit dem Übersetzen (Transkription) der Erbinformation (DNA zu mRNA) im Zellkern beginnt und mit der Biosynthese (Translation) von Proteinen im Zellplasma endet. Obwohl die Expression einzelner Gene sehr stark reguliert ist, ist der Grad der Genexpression in genetisch identischen Zellen nicht gleich. Diese Heterogenität resultiert aus einer Kombination von äußeren Einflüssen und der zufälligen Variation (Stochastizität), die allen biochemischen Prozessen innewohnt. Die Variabilität von Genexpression wurde als eine wesentliche Ursache für phenotypische Unterschiede zwischen genetisch identischen Individuen identifiziert. Welche Rolle sie aber in der generellen Regulation von Genexpression spielt ist weitgehend unverstanden.
Lay summary
Inhalt und Ziele des Forschungsprojekts:

Das Hauptziel dieses Projekts besteht in der Entwicklung einer Fluoreszenzmikroskopie-basierten Methode, die es gleichzeitig erlaubt die Translation und den Abbau individueller mRNA Moleküle in lebenden Zellen zu beobachten. Mithilfe dieser Technologie wollen wir grundlegende biologische Fragen beantworten und z.B. messen wie oft eine einzelne mRNA abgelesen werden kann, bevor sieht abgebaut wird. Weiterhin werden wir die Methode dazu nutzen um den Zusammenhang zwischen post-transkriptionellen Regulationsmechanismen (z.B. RNA-Interferenz) und der Variabilität von Genexpression zu erforschen.


Kontext:

Wie viel Protein aus jedem einzelnen mRNA Molekül hergestellt wird, hängt von der Transkriptions-, Translations- und Abbaurate jeder einzelnen mRNA ab. Bisher war es technisch nicht möglich, die Translations- und Abbauraten einzelner mRNAs zu bestimmen. Daher war es auch nicht möglich Genexpression hinsichtlich ihrer lokalen Variabilität innerhalb der Zelle zu charakterisieren bzw. auf diese Weise den Einfluss von kleinen Veränderungen im Ausmaß der Genexpression zu verstehen. Bei diesem Projekt handelt sich um ein Grundlagenforschungsprojekt, dessen Ziel es ist eine neue Methode zu entwickeln, die es erlauben wird grundlegende biologische Fragestellungen zur zeitlich-lokalen Regulation von Genexpression zu beantworten.
Direct link to Lay Summary Last update: 10.01.2017

Responsible applicant and co-applicants

Employees

Publications

Publication
Detection of the First Round of Translation: The TRICK Assay.
Voigt Franka, Eglinger Jan, Chao Jeffrey A. (2018), Detection of the First Round of Translation: The TRICK Assay., in Gaspar Imre (ed.), Springer New York, New York, NY, 373-384.
Single-Molecule Quantification of Translation-Dependent Association of mRNAs with the Endoplasmic Reticulum
Voigt Franka, Zhang Hui, Cui Xianying A., Triebold Desiree, Liu Ai Xin, Eglinger Jan, Lee Eliza S., Chao Jeffrey A., Palazzo Alexander F. (2017), Single-Molecule Quantification of Translation-Dependent Association of mRNAs with the Endoplasmic Reticulum, in CELL REPORTS, 21(13), 3740-3753.
The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells.
Hovathova Ivana, Voigt Franka, Kotrys Anna V., Zhan Yinxiu, Artus-Revel Caroline G., Eglinger Jan, Stadler Michael B., Giorgetti Luca, Chao Jeffrey A. (2017), The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells., in Molecular Cell, 68(3), 615-625.
Quantification of mRNA Turnover in Living Cells: A Pipeline for TREAT Data Analysis
VoigtFranka, EglingerJan, ChaoJeffrey A., Quantification of mRNA Turnover in Living Cells: A Pipeline for TREAT Data Analysis, in Shav-Tal Yaron (ed.), Springer Nature, Humana Press, New York.

Collaboration

Group / person Country
Types of collaboration
Palazzo lab/University of Toronto Canada (North America)
- 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
RNA 2019 - Annual Meeting of the RNA Society Poster Assessing translation and decay dynamics via live-cell imaging of single mRNAs in different subcellular localizations 11.06.2019 Krakow, Poland Voigt Franka;
Austrian Swiss RNA Meeting Poster Assessing translation and decay dynamics via live-cell imaging of single mRNAs in different subcellular localizations 31.01.2019 Salzburg, Austria Voigt Franka;
The Complex Life of RNA Poster Assessing translation and decay dynamics via live-cell imaging of single mRNAs in different subcellular localizations 03.10.2018 EMBL Heidelberg, Germany Voigt Franka;
RNA 2017 - Annual Meeting of the RNA Society Poster Single-molecule quantification of translation-dependent association of mRNAs with the endoplasmic reticulum 30.05.2017 Prague, Czech Republic Voigt Franka;


Associated projects

Number Title Start Funding scheme
182314 Spatiotemporal regulation of stress-dependent translation 01.10.2018 Project funding (Div. I-III)

Abstract

Biological systems are intrinsically noisy. Gene expression variability, or “noise”, originates from a combination of externally caused fluctuations and the internal stochasticity of all biochemical processes involved. It has been identified as a major source of phenotypic variability between genetically identical individuals. The amount of protein generated from expression of a gene depends on the rates of mRNA transcription, translation and decay. While single-molecule methods have revealed insights into the dynamics of mRNA transcription, it has not been possible to investigate the variability of mRNA translation or degradation.We have partially overcome this limitation by designing the TREAT (3(Three) RNA End Accumulation during Turnover) reporter that can monitor the degradation of individual mRNAs in living cells via stabilization of degradation intermediates using viral pseudo knots. Several recent publications have described novel imaging- based techniques (e.g. SunTag imaging) that can directly observe nascent polypeptides as they are synthesized on individual mRNAs. Here, I propose to develop a fluorescent biosensor that will enable simultaneous single- molecule measurements of mRNA translation (SunTag) and degradation (TREAT) in living cells. I will apply it to directly measure how many times and how efficiently transcripts are translated before their degradation as well as to investigate what translational efficiency depends on (i.e. subcellular localization, cell cycle phase). Posttranscriptional gene regulatory mechanisms, such as small RNA pathways, have been proposed to regulate gene expression via reduction of gene expression noise. At the same time, mammalian microRNA (miRNA) regulation has been reported to predominantly function via destabilization of mRNA transcripts. To understand how miRNA-mediated regulation manipulates gene expression in detail, I propose to apply my reporter to quantify and correlate the contributions of transcription, translation and degradation to total intrinsic gene expression noise of miRNA site-containing transcripts. I will use a combination of single-RNA fluorescence in-situ hybridization (FISH) and immunofluorescence experiments to investigate the dynamics of transcription, translation and turn-over in single fixed cells as well as live-cell imaging experiments to assess their variability on individual transcripts.
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