Absolute quantitative measurement of transcriptional kinetic parameters in vivo.
Absolute quantitative measurement of transcriptional kinetic parameters in vivo.
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DOI：
10.1093/nar/gkw596
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发表时间：
2016-10-14
影响因子：
14.9
通讯作者：
Kim M
中科院分区：
文献类型：
--
作者：
Iyer S;Park BR;Kim M
mRNA的表达包括转录起始、延伸和降解。在细胞中，这些动态过程受到高度调控。然而，由于缺乏能够直接测量的方法，体内动态过程的实验表征是困难的。我们提出了一种高度敏感和通用的方法，可以直接表征动态过程。我们的方法是基于单分子荧光原位杂交（smFISH）和杂交信号的定量分析。我们将多个标记有不同光谱荧光团的探针杂交到单个mrna的多个亚区，并可视化这些亚区合成和降解的动力学。数据的定量分析导致mRNA诱导滞后时间（外部信号激活转录起始所需的时间）、转录起始率、转录延伸速度（即mRNA链生长速度）、转录物过早终止率和降解率的绝对量化。将我们的方法应用于三个不同的生物学问题，我们证明了我们的方法如何适用于揭示以前难以研究的mRNA表达动力学。我们期望这种绝对定量可以极大地促进对基因表达及其在转录起始、延伸和降解水平上的调控的理解。
mRNA expression involves transcription initiation, elongation and degradation. In cells, these dynamic processes are highly regulated. However, experimental characterization of the dynamic processes in vivo is difficult due to the paucity of methods capable of direct measurements. We present a highly sensitive and versatile method enabling direct characterization of the dynamic processes. Our method is based on single-molecule fluorescence in situ hybridization (smFISH) and quantitative analyses of hybridization signals. We hybridized multiple probes labelled with spectrally distinct fluorophores to multiple sub-regions of single mRNAs, and visualized the kinetics of synthesis and degradation of the sub-regions. Quantitative analyses of the data lead to absolute quantification of the lag time of mRNA induction (the time it takes for external signals to activate transcription initiation), transcription initiation rate, transcription elongation speed (i.e. mRNA chain-growth speed), the rate of premature termination of transcripts and degradation rates. Applying our method to three different biological problems, we demonstrated how our method may be applicable to reveal dynamics of mRNA expression that was difficult to study previously. We expect such absolute quantification can greatly facilitate understanding of gene expression and its regulation working at the levels of transcriptional initiation, elongation and degradation.