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

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      ACS Central Science
      American Chemical Society

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          Abstract

          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.

          Abstract

          We developed a method to image and quantify changes in mRNA translation in fixed cells. The method utilizes RNA in situ hybridization and the hybridization chain reaction to produce bright fluorescence when ribosomes are bound to an mRNA of interest.

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          Most cited references31

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          Translational control of long-lasting synaptic plasticity and memory.

          Long-lasting forms of synaptic plasticity and memory are dependent on new protein synthesis. Recent advances obtained from genetic, physiological, pharmacological, and biochemical studies provide strong evidence that translational control plays a key role in regulating long-term changes in neural circuits and thus long-term modifications in behavior. Translational control is important for regulating both general protein synthesis and synthesis of specific proteins in response to neuronal activity. In this review, we summarize and discuss recent progress in the field and highlight the prospects for better understanding of long-lasting changes in synaptic strength, learning, and memory and implications for neurological diseases.
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            Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics

            S.-E. Ong (2002)
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              Correlation between Protein and mRNA Abundance in Yeast

              We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeastSaccharomyces cerevisiaegrowing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.
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                Author and article information

                Journal
                ACS Cent Sci
                ACS Cent Sci
                oc
                acscii
                ACS Central Science
                American Chemical Society
                2374-7943
                2374-7951
                03 May 2017
                24 May 2017
                : 3
                : 5
                : 425-433
                Affiliations
                [1]Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States
                Author notes
                Article
                10.1021/acscentsci.7b00048
                5445550
                28573204
                9d485b05-1d55-40e3-9c14-3ec38a058316
                Copyright © 2017 American Chemical Society

                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.

                History
                : 25 January 2017
                Categories
                Research Article
                Custom metadata
                oc7b00048
                oc-2017-000489

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