Burke Kelly S, Antilla Katie A, Tirrell David A
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, United States.
ACS Cent Sci. 2017 May 24;3(5):425-433. doi: 10.1021/acscentsci.7b00048. Epub 2017 May 3.
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.
单分子荧光原位杂交（smFISH）是一种简单且广泛应用的方法，用于测量单细胞中mRNA转录本的丰度和定位。一种用于测量mRNA翻译的类似单分子原位方法将有助于更全面地理解基因调控。在此，我们描述一种荧光测定法，用于检测核糖体与mRNA的相互作用（FLARIM）。该方法采用smFISH来可视化和表征固定细胞中mRNA单分子的翻译。为了可视化核糖体 - mRNA相互作用，我们使用一对寡核苷酸探针，它们分别与核糖体（通过rRNA）和感兴趣的mRNA结合，并且当探针紧密接近时，通过杂交链式反应（HCR）产生强烈的荧光信号。FLARIM不需要基因操作，几乎适用于任何内源性mRNA转录本，并提供空间和时间信息。我们证明，在用嘌呤霉素抑制全局翻译后，FLARIM对核糖体与mRNA结合的变化敏感。我们还表明，FLARIM检测到核糖体与一种mRNA结合的变化，该mRNA的翻译会因铁浓度增加而上调。
