A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing .Ribosome mRNA Interactions in Single Cells
A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing .Ribosome mRNA Interactions in Single Cells
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DOI：
10.1021/acscentsci.7b00048
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发表时间：
2017-05-01
影响因子：
18.2
通讯作者：
Tirrell, David A.
中科院分区：
文献类型：
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作者：
Burke, Kelly S.;Antilla, Katie A.;Tirrell, David A.
单分子荧光原位杂交(SmFISH)是一种简单、广泛应用于单细胞内mRNA转录丰度和定位的方法。一种可比拟的单分子原位方法来测量mRNA的翻译将使我们能够更全面地了解基因调控。在这里，我们描述了一种检测核糖体与mRNA相互作用的荧光分析方法(FLARIM)。该方法采用smFISH来可视化和表征固定细胞中单分子信使核糖核酸的翻译。为了可视化核糖体-mRNA的相互作用，我们使用了一对寡核苷酸探针，它们分别与核糖体(通过rRNA)和目标mRNA结合，当探针接近时，它们通过杂交链式反应(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FLARIM对核糖体与mRNA结合的变化是敏感的，当用嘌呤霉素抑制整体翻译时。我们还表明，FLARIM检测到核糖体与mRNA关联的变化，其翻译随着铁浓度的增加而上调。
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.