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Messenger RNA regulation by 5'-cap structure

5帽结构对信使RNA的调控

基本信息

批准号：
14035104
负责人：
MIZUMOTO Kiyohisa
金额：
$ 40.45万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14035104/
关键词：
cap structure RNA polvmerase II RNA-dependent RNA polymerase Sendai virus Influenza virus mRNA surveillance aystem translation repression プロテアソームキャッピングノンストップmRNA ポリ(A)鎖転写開始 mRNA分解制御キャッピング酵素転写複合体 RNA依存RNA合成酵素 RNAポリメラーゼI

项目摘要

(A) Enzyme mechanisms of mRNA cap formation in eukaryotes as well as negative-strand RNA viruses have been studied. 1) We have shown that the phosphorylation of RNA polymerase II carboxyl terminal domain (CTD) by TFIIH-associated CTD kinase (CAK) is important for successful capping of nascent RNA chain. Using an in vitro transcription system with immobilized template, we demonstrated that capping occurs when the nascent RNA chain grows to 18-19 nt in length. 2) We demonstrated that SeV RNA-dependent RNA polymerase (RdRP) L protein catalyzes cap methylation of virus specific mRNA. This is to our knowledge the first direct biochemical evidence to show that mononegavirus L protein catalyszes cap G-7-methylation. 3) The influenza virus (FluV) RdRP exhibits a cap-dependent endonuclease activity, which cleaves host mRNAs to produce capped RNA fragments with 11 to13 nucleotides. The resulting capped RNA fragments serve as a primer to initiate viral mRNA synthesis. We found that Flu-A and -B endonucleases exhibited different substrate specificities with regard to the extent as well as the position of cap methylation.(B) A novel mRNA surveillance for mRNA lacking a termination codon (nonstop mRNA) has been proposed in which Ski7p is thought to recognize stalled ribosomes at the 3' end of mRNA. We found that the level of protein product of nonstop mRNA containing a poly(A) tail was reduced 100-fold, and this reduction was due to rapid mRNA degradation, translation repression and protein destabilization, at least in part, by the proteasome. Insertion of a poly(A) tract upstream of a termination codon resulted in translation repression and protein destabilization but not rapid mRNA decay. We propose that translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization.

(A)已经研究了真核生物和负链RNA病毒中mRNA帽子形成的酶机制。1)我们已经证明，TFIIH相关的CTD激酶(CAK)对RNA聚合酶II羧基末端结构域(CTD)的磷酸化对于成功封顶新生RNA链是重要的。使用固定化模板的体外转录系统，我们证明了当新生的RNA链长到18-19个核苷酸时，就会发生封顶。2)我们证明了SeVRNA依赖的RNA聚合酶L蛋白能催化病毒特异性基因的帽甲基化。据我们所知，这是第一个直接的生化证据表明，MononegaVirus L蛋白催化CAP G-7甲基化。3)流感病毒(FluV)RdRP具有帽依赖的内切酶活性，能裂解宿主的mRNAs，产生11~13个核苷酸的帽状RNA片段。由此产生的有帽的RNA片段作为启动病毒信使核糖核酸合成的引子。我们发现，Flu-A和Flu-B内切酶在帽甲基化的程度和位置上表现出不同的底物特异性。(B)提出了一种新的对缺少终止密码子的mRNA的mRNA监测方法，其中Ski7p被认为识别mRNA 3‘端的停滞核糖体。我们发现，含有Poly(A)尾巴的不间断mRNA的蛋白质产物水平降低了100倍，这是由于蛋白质酶体快速的mRNA降解、翻译抑制和蛋白质失稳，至少部分是由于蛋白质酶体。在终止密码子的上游插入Poly(A)区会导致翻译抑制和蛋白质失稳，但不会导致快速的mRNA衰退。我们认为，Poly(A)尾巴的翻译通过翻译抑制和蛋白质失稳在不间断的mRNA监测中发挥关键作用。