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Involvement of mRNA capping enzyme in transcription initiation

mRNA 加帽酶参与转录起始

基本信息

批准号：
14580630
负责人：
SHIBAGAKI Yoshio
金额：
$ 2.24万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580630/
关键词：
Capping enzyme C-terminal domain RNA polymerase II in vitro transcription Transcription factor Protein phosphorylation

项目摘要

The mRNA capping occurs soon after transcription initiation and before other processing events. The several mRNA processing factors are coupled to transcription through binding to the C-terminal domain of RNA polymerase II (CTD). mRNA-capping enzyme directly binds to the phosphorylated CTD, and catalyzes the co-transcriptional cap formation. However, the precise mechanism of the involvement of capping enzyme in pol II transcription has been unclear.(1) To examine the involvement of capping enzymes in pol II transcription, we developed in vitro transcription/capping system using immobilized DNA template. Immobilized templates were incubated with HeLa nuclear extract to form initiation complex (PICs) on promoter region. After washing to remove unbound proteins, PICs were subjected to elongation reaction in the presence of 3'-O-methly CTP in order to stall elongation complex at the first C residue. Using this system, we found that capping occurs in 18-19 nt RNA length in the case of adenovirus major late promoter.(2) To investigate the functional interaction of capping enzyme and pol II, we subjected highly purified calf thymus pol II to capping assay with various kinds of CTD kinases. Capped RNAs were separated by Boronate-affinity PAGE (BAGE) to evaluate capping efficiency. We found that non-phosphorylated pol II stimulated capping activity about 2.5-fold. Furthermore, CAK phosphorylated pol II increased in capping activity about 1.8-fold compared with non-phosphorylated pol II. These results suggest that the CAK phosphorylation of pol II CTD is important for activation of capping enzyme and other portion of pol II also may involve in this activation.

mRNA 加帽发生在转录起始后不久和其他处理事件之前。几种 mRNA 加工因子通过与 RNA 聚合酶 II (CTD) 的 C 端结构域结合而与转录偶联。 mRNA加帽酶直接与磷酸化的CTD结合，并催化共转录帽的形成。然而，加帽酶参与pol II转录的确切机制尚不清楚。(1)为了研究加帽酶参与pol II转录，我们利用固定化DNA模板开发了体外转录/加帽系统。固定化模板与 HeLa 核提取物一起孵育，在启动子区域形成起始复合物 (PIC)。洗涤去除未结合的蛋白质后，PIC 在 3'-O-甲基 CTP 存在下进行延伸反应，以在第一个 C 残基处阻止延伸复合物。利用该系统，我们发现腺病毒主要晚期启动子在18-19 nt RNA长度处发生加帽。(2)为了研究加帽酶和pol II的功能相互作用，我们对高纯度的小牛胸腺pol II进行了各种CTD激酶的加帽实验。通过硼酸亲和 PAGE (BAGE) 分离加帽的 RNA 以评估加帽效率。我们发现非磷酸化的 pol II 刺激加帽活性约 2.5 倍。此外，与非磷酸化的pol II相比，CAK磷酸化的pol II的加帽活性增加了约1.8倍。这些结果表明，pol II CTD 的 CAK 磷酸化对于加帽酶的激活很重要，并且 pol II 的其他部分也可能参与这种激活。