Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene.
Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene.
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DOI：
10.1038/s41467-021-23417-0
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发表时间：
2021-05-26
影响因子：
16.6
通讯作者：
Stasevich TJ
中科院分区：
文献类型：
--
作者：
Forero-Quintero LS;Raymond W;Handa T;Saxton MN;Morisaki T;Kimura H;Bertrand E;Munsky B;Stasevich TJ
RNA聚合酶II（RNAP 2）的羧基末端结构域在真核细胞中的转录期间被磷酸化。虽然残留物特异性磷酸化已被映射与精致的空间分辨率沿着的1D基因组在一个群体中的固定细胞使用免疫沉淀为基础的测定，磷酸化的时间，动力学和空间组织沿着一个单拷贝基因尚未在活细胞中测量。在这里，我们通过将多色，单分子显微镜与基于荧光抗体的探针相结合来实现这一目标，这些探针特异性地结合活细胞中内源性RNAP 2的不同磷酸化形式。将这种方法应用于单拷贝HIV-1报告基因提供了活细胞证据，证明RNAP 2沿着基因长度分布的异质性，以及丝氨酸5磷酸化RNAP 2簇在空间和时间上与新生mRNA合成保持分离。计算模型确定，在典型的转录爆发期间，5至40个RNAP 2聚集在启动子周围，其中大多数在到达后6秒内在丝氨酸5处磷酸化，大约一半在约1.5分钟内逃离启动子。总之，我们的数据提供了活细胞支持的概念，瞬时形成启动子周围的高效转录簇，并含有高浓度的RNAP 2磷酸化丝氨酸5。在转录过程中，RNA聚合酶II（RNAP 2）被募集到启动子并逐步磷酸化;到目前为止，这些步骤在活细胞中的单拷贝基因中还没有可视化。在这里，作者使用单分子显微镜观察活细胞中单个位点的内源性磷酸化RNAP 2和新生mRNA合成。
The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5. During transcription, RNA polymerase II (RNAP2) is recruited to promoters and phosphorylated stepwise; so far, these steps have not been visualized in a single-copy gene in live cells. Here the authors use single-molecule microscopy to visualize endogenous phosphorylated RNAP2 and nascent mRNA synthesis at a single locus in living cells.
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影响因子：
4.6
作者：
Cho WK;Jayanth N;Mullen S;Tan TH;Jung YJ;Cissé II
通讯作者：
Cissé II
影响因子：
7.7
作者：
Cho, Won-Ki;Jayanth, Namrata;Cisse, Ibrahim I.
通讯作者：
Cisse, Ibrahim I.
影响因子：
2.9
作者：
Carlini, Lina;Benke, Alexander;Manley, Suliana
通讯作者：
Manley, Suliana
影响因子：
9.8
作者：
Brody Y;Neufeld N;Bieberstein N;Causse SZ;Böhnlein EM;Neugebauer KM;Darzacq X;Shav-Tal Y
通讯作者：
Shav-Tal Y
DOI：
10.1126/science.aao3136
发表时间：
2018-03-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gu B;Swigut T;Spencley A;Bauer MR;Chung M;Meyer T;Wysocka J
通讯作者：
Wysocka J