Messenger RNA regulation by 5'-cap structure

(A)对真核生物和负链RNA病毒中mRNA帽形成的酶机制进行了研究。1)我们已经证明，tfih相关的CTD激酶（CAK）对RNA聚合酶II羧基末端结构域（CTD）的磷酸化对于新生RNA链的成功盖帽至关重要。利用固定化模板的体外转录系统，我们证明了当新生RNA链长到18-19 nt时发生了封顶。2)我们证明了SeV RNA依赖的RNA聚合酶（RdRP） L蛋白催化病毒特异性mRNA的帽甲基化。据我们所知，这是第一个直接的生化证据，表明单核病毒L蛋白催化帽g -7甲基化。3)流感病毒（FluV） RdRP具有帽依赖的核酸内切酶活性，可切割宿主mrna，产生11 ~ 13个核苷酸的带帽RNA片段。由此产生的带帽RNA片段作为引物启动病毒mRNA合成。我们发现流感- a和-B内切酶在帽甲基化的程度和位置方面表现出不同的底物特异性。(B)对缺乏终止密码子的mRNA（不间断mRNA）提出了一种新的mRNA监视方法，其中Ski7p被认为可以识别mRNA 3'端停滞的核糖体。我们发现含有聚(a)尾的不间断mRNA的蛋白产物水平降低了100倍，这种降低是由于mRNA的快速降解，翻译抑制和蛋白质不稳定，至少部分是由蛋白酶体造成的。在终止密码子上游插入多聚a链导致翻译抑制和蛋白质不稳定，但不会导致mRNA的快速衰变。我们认为poly(A)尾部的翻译通过翻译抑制和蛋白质不稳定在不间断的mRNA监视中起着至关重要的作用。

(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.