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