Live imaging of transcription sites using an elongating RNA polymerase II-specific probe.
Live imaging of transcription sites using an elongating RNA polymerase II-specific probe.
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使用拉长RNA聚合酶II特异性探针对转录位点的实时成像。
DOI：
10.1083/jcb.202104134
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发表时间：
2022-02-07
期刊：
影响因子：
--
通讯作者：
Kimura H
中科院分区：
文献类型：
--
作者：
Uchino S;Ito Y;Sato Y;Handa T;Ohkawa Y;Tokunaga M;Kimura H
内野等人。开发了一种基因编码探针，可特异性检测活细胞中 Ser2 磷酸化、延伸形式的 RNA 聚合酶 II。 Ser2 磷酸化 RNA 聚合酶焦点比染色质复制域和富含 p300 的焦点表现出更动态的运动。在真核细胞核中，大多数基因由 RNA 聚合酶 II (RNAP2) 转录，RNA 聚合酶 II 的调控是了解基因组和细胞功能的关键。 RNAP2 具有长七肽重复序列 (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7)，并且 Ser2 以延伸形式磷酸化。为了检测活细胞中的 RNAP2 Ser2 磷酸化 (RNAP2 Ser2ph)，我们开发了一种基因编码的修饰特异性细胞内抗体 (mintbody) 探针。 RNAP2 Ser2ph-mintbody 表现出许多焦点，可能代表转录“工厂”，并且焦点在有丝分裂期间和 Ser2 激酶抑制剂中减少。使用磷酸肽的体外结合测定证实了薄荷体的特异性。与参与起始的因素相比，RNAP2 Ser2ph-mintbody 焦点与与延长 RNAP2 相关的蛋白质共定位。这些结果支持薄荷体定位代表活细胞中 RNAP2 Ser2ph 位点的观点。 RNAP2 Ser2ph-mintbody 焦点显示出与染色质类似的受限扩散运动，但它们比 DNA 复制结构域和富含 p300 的焦点更具移动性，表明伸长的 RNAP2 复合物与更受限的染色质结构域分离。
Uchino et al. have developed a genetically encoded probe to specifically detect the Ser2-phosphorylated, elongating form of RNA polymerase II in living cells. Ser2-phosphorylated RNA polymerase foci exhibit more dynamic motion than chromatin replication domains and p300-enriched foci. In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody’s specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.
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影响因子：
64.8
作者：
Heintzman, Nathaniel D.;Hon, Gary C.;Hawkins, R. David;Kheradpour, Pouya;Stark, Alexander;Harp, Lindsey F.;Ye, Zhen;Lee, Leonard K.;Stuart, Rhona K.;Ching, Christina W.;Ching, Keith A.;Antosiewicz-Bourget, Jessica E.;Liu, Hui;Zhang, Xinmin;Green, Roland D.;Lobanenkov, Victor V.;Stewart, Ron;Thomson, James A.;Crawford, Gregory E.;Kellis, Manolis;Ren, Bing
通讯作者：
Ren, Bing
影响因子：
7.7
作者：
Cho, Won-Ki;Jayanth, Namrata;Cisse, Ibrahim I.
通讯作者：
Cisse, Ibrahim I.
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
影响因子：
5.6
作者：
Francette AM;Tripplehorn SA;Arndt KM
通讯作者：
Arndt KM
影响因子：
64.8
作者：
Guo, Yang Eric;Manteiga, John C.;Young, Richard A.
通讯作者：
Young, Richard A.