Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, State Key Laboratory of Genetic Engineering, Department of Biochemistry and Biophysics, School of Life Sciences, Shanghai Key Laboratory of Radiation Oncology, and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai 200032, China; The International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, China, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China; Human Phenome Institute, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200433, China.
Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, State Key Laboratory of Genetic Engineering, Department of Biochemistry and Biophysics, School of Life Sciences, Shanghai Key Laboratory of Radiation Oncology, and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai 200032, China.
Cell Rep. 2022 Apr 26;39(4):110732. doi: 10.1016/j.celrep.2022.110732.
RNA polymerase II (Pol II)-mediated transcription in metazoans requires precise regulation. RNA Pol II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). Here, we show that RPAP2 binds hypo-/hyper-phosphorylated Pol II with undetectable phosphatase activity. The structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents and disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in inhibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating a critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to inhibit PIC assembly and transcription initiation and suggests a transcription checkpoint.
真核生物中 RNA 聚合酶 II（Pol II）介导的转录需要精确的调控。RNA Pol II 相关蛋白 2（RPAP2）先前被鉴定为将 Pol II 从细胞质运输到细胞核，并使 Pol II C 端结构域（CTD）去磷酸化。在这里，我们表明 RPAP2 与低/高磷酸化的 Pol II 结合，但无检测到的磷酸酶活性。RPAP2-Pol II 的结构由于三个空间位阻的存在，显示了 RPAP2-Pol II 和起始前复合物（PIC）的互斥组装。RPAP2 阻止并破坏了 Pol II-TFIIF 的相互作用，并损害了体外转录起始，表明其在抑制 PIC 组装中具有功能。细胞中 RPAP2 的缺失导致启动子处 TFIIF 和 Pol II 的全局积累，表明 RPAP2 在抑制 PIC 组装中具有关键作用，而与其假定的磷酸酶活性无关。我们的研究表明，RPAP2 作为一种门控因子抑制 PIC 组装和转录起始，并提示存在转录检查点。
