Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II
Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II
复制标题
DOI：
10.1101/2021.04.27.441582
复制
发表时间：
2021-04
期刊：
影响因子：
--
通讯作者：
Satoshi Uchino;Yuma Ito;Y. Sato;Tetsuya Handa;Y. Ohkawa;M. Tokunaga;H. Kimura
中科院分区：
文献类型：
--
作者：
Satoshi Uchino;Yuma Ito;Y. Sato;Tetsuya Handa;Y. Ohkawa;M. Tokunaga;H. Kimura
在真核生物的细胞核中，大多数基因是由RNA聚合酶II （RNAP2）转录的。RNAP2在细胞核中的转录调控是理解基因组和细胞功能的关键。RNAP2的最大亚基在c端结构域有一个很长的七肽重复序列（Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7）， Ser2在RNAP2的延伸形式上被磷酸化。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修饰特异性细胞内抗体（mintbody）探针。RNAP2 Ser2ph-mintbody探针显示了许多灶，可能代表了活HeLa细胞中的转录“工厂”，当用雷公藤甲素诱导RNAP2降解和黄酮吡醇抑制Ser2ph处理细胞时，灶减少。使用磷酸肽的体外结合试验证实了薄荷体的ser2ph特异性结合。这些结果支持了薄荷体定位代表活细胞中RNAP2 Ser2ph位点的观点。与参与转录起始位点周围转录激活的因子（如CDK9和BRD4）相比，RNAP2 Ser2ph-mintbody病灶与与延长RNAP2相关的蛋白（如CDK12和Paf1复合物组分）共定位。跟踪分析显示，RNAP2 Ser2ph-mintbody区域与染色质一样具有受限的扩散运动，但与常染色质区域相比更具移动性，这表明拉长的RNAP2复合物与更受限的起始簇分离。作者开发了一种基因编码探针，用于特异性检测活细胞中Ser2磷酸化的RNA聚合酶II的伸长形式。Ser2磷酸化聚合酶的运动比染色质结构域更动态，这表明拉长的复合物与更受限制的起始簇分开。
In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. 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 probe exhibited numerous foci, possibly representing transcription “factories” in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters. Summary The authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.