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A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells

Burke, Kelly S. and Antilla, Katie A. and Tirrell, David A. (2017) A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells. ACS Central Science, 3 (5). pp. 425-433. ISSN 2374-7943. PMCID PMC5445550. doi:10.1021/acscentsci.7b00048.

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Single-molecule fluorescence in situ hybridization (smFISH) is a simple and widely used method to measure mRNA transcript abundance and localization in single cells. A comparable single-molecule in situ method to measure mRNA translation would enable a more complete understanding of gene regulation. Here we describe a fluorescence assay to detect ribosome interactions with mRNA (FLARIM). The method adapts smFISH to visualize and characterize translation of single molecules of mRNA in fixed cells. To visualize ribosome–mRNA interactions, we use pairs of oligonucleotide probes that bind separately to ribosomes (via rRNA) and to the mRNA of interest, and that produce strong fluorescence signals via the hybridization chain reaction (HCR) when the probes are in close proximity. FLARIM does not require genetic manipulation, is applicable to practically any endogenous mRNA transcript, and provides both spatial and temporal information. We demonstrate that FLARIM is sensitive to changes in ribosome association with mRNA upon inhibition of global translation with puromycin. We also show that FLARIM detects changes in ribosome association with an mRNA whose translation is upregulated in response to increased concentrations of iron.

Item Type:Article
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URLURL TypeDescription Information CentralArticle
Burke, Kelly S.0000-0002-3615-9828
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2017 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: January 25, 2017; Published: May 3, 2017. We thank the National Science Foundation Graduate Research Fellowship Program (NSF GRFP, Grant Number 1144469), the Rose Hills Foundation (Caltech SURF), the German Research Foundation (Grant No. MI 1315/4), and the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation for support of this work. We thank H. M. T. Choi and N. A. Pierce for suggesting the use of a linker probe carrying an HCR initiator as a mechanism for selectively generating signal from cognate probe pairs colocalized by targets in the sample. We thank Florian Mueller for development of FISH-quant and the Broad Institute for development of Cell Profiler for image analysis. We thank Johannes Stegmaier and the Center for Advanced Methods in Biological Image Analysis at the Beckman Institute (CAMBIA) for adapting the XPIWIT software tool for analysis of nuclear transcripts. We thank Andres Collazo for assistance with the LSM 800 confocal microscope in the Biological Imaging Facility of the Beckman Institute at Caltech.
Funding AgencyGrant Number
NSF Graduate Research FellowshipDGE-1144469
Rose Hills FoundationUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)MI 1315/4
Gordon and Betty Moore FoundationUNSPECIFIED
Issue or Number:5
PubMed Central ID:PMC5445550
Record Number:CaltechAUTHORS:20170504-090316077
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Official Citation:A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells Kelly S. Burke, Katie A. Antilla, and David A. Tirrell ACS Central Science 2017 3 (5), 425-433 DOI: 10.1021/acscentsci.7b00048
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:77188
Deposited By: Tony Diaz
Deposited On:04 May 2017 16:11
Last Modified:28 Mar 2022 22:00

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