Quantification of mRNA translation in live cells using single-molecule imaging

TY - JOUR

T1 - Quantification of mRNA translation in live cells using single-molecule imaging

AU - Khuperkar, Deepak

AU - Hoek, Tim A

AU - Sonneveld, Stijn

AU - Verhagen, Bram M P

AU - Boersma, Sanne

AU - Tanenbaum, Marvin E

PY - 2020/4

Y1 - 2020/4

N2 - mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated).

AB - mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated).

KW - HEK293 Cells

KW - Humans

KW - Image Processing, Computer-Assisted/methods

KW - Protein Biosynthesis/genetics

KW - RNA, Messenger/analysis

KW - Single Molecule Imaging/methods

U2 - 10.1038/s41596-019-0284-x

DO - 10.1038/s41596-019-0284-x

M3 - Article

C2 - 32076351

SN - 1754-2189

VL - 15

SP - 1371

EP - 1398

JO - Nature Protocols

JF - Nature Protocols

IS - 4

ER -