Cap specific N6 methylation of viral mRNA by the cellular methyltransferase PCIF1

该项目的长期目标是确定存在于真核细胞mRNAs转录起始点的帽特异性N6，2‘-O-二甲基腺苷(M6AM)的功能(S)。我们和其他人最近发现了负责N6位帽子-近端腺苷甲基化的细胞mRNA甲基转移酶是磷酸化的羧基末端结构域相互作用因子1(PCIF1)。PCIF1结合宿主RNA聚合酶II的磷酸化C-末端结构域，选择性地修饰帽近端A，随后鸟嘌呤-N7-甲基酶和核糖-2‘-O甲基酶对帽结构进行甲基化。帽-近端m6Am修饰的功能意义尚不确定，已发表的文献对mRNA的稳定性和翻译得出了不同的结论。在细胞质中复制的病毒，如负链RNA病毒水泡性口炎病毒(VSV)，尽管没有病毒编码的N6，2‘-O-二甲基转移酶，但也含有对感染细胞合成的mRNA的这种帽-近端m6Am修饰。在初步的数据中，我们发现PCIF1在VSV感染细胞中被重新定位到细胞质中，并甲基化VSV mRNA。5个VSV mRNAs的特征很好，我们已经开发了必要的工具来定义m6Am如何影响每个mRNAs的功能。我们的初步数据表明，m6Am的缺失既不影响m6Am的mRNA稳定性，也不影响m6Am的翻译，在培养的293T和Hela细胞中，在基础条件下病毒复制没有影响。然而，用干扰素对细胞进行预处理表明，PCIF1的缺失会进一步抑制病毒mRNA的翻译，并更显著地抑制病毒的生长。这种依赖PCIF1的表型表明，这种帽-近端m6Am的一个功能是区分自我和非自我mRNA。当PCIF1第一次被检测到时，mRNA帽被假设是随着真核生物的进化而出现的，它取代了Shine-Dalgarno序列，用于将核糖体定向到mRNAs，并保护mRNAs不被5‘外源核糖核酸酶消化，从而提供了一种区分自体和外源mRNAs的早期方法。现有的病毒很可能是在这种RNA甲基化的情况下进化的，以逃避真核生物的自卫系统。为了进一步探讨PCIF1修饰病毒RNA的作用，我们建立了一只PCIF1-/-小鼠，提供了一种额外的独特试剂来从机械上剖析病毒mRNA的m6Am在体内的作用。利用这些初步数据，我们将在细胞培养和体内使用遗传学、生化、细胞生物学和病毒学方法来剖析m6Am和PCIF1介导的mRNA甲基化的作用。我们的基本假设是，PCIF1对信使核糖核酸的修饰有助于区分自我和非自我信使核糖核酸，病毒已经选择了PCIF1来确保有效的复制。

The long-term goal of this project is to define the function(s) of the cap-specific N6, 2'-O-dimethyladenosine (m6 Am) present at the transcription start site of eukaryotic mRNAs. We and others recently identified the cellular mRNA methyltransferase responsible for methylation of the cap-proximal adenosine at the N6 position as phosphorylated carboxy-terminal domain interacting factor 1 (PCIF1). PCIF1 binds the phosphorylated C- terminal domain of host RNA polymerase II to selectively modify the cap proximal A, following the sequential methylation of the cap-structure by the guanine-N7-methylase and ribose-2'-O methylase. The functional significance of the cap-proximal m6Am modification is uncertain with published literature reaching different conclusions regarding mRNA stability and translation. Viruses that replicate in the cytoplasm such as the negative-strand RNA virus vesicular stomatitis virus (VSV) also contain this cap-proximal m6Am modification on mRNA synthesized in infected cells despite the absence of a viral encoded N6, 2'-O-dimethyltransferase. In preliminary data we have found that PCIF1 is relocalized to the cytoplasm in VSV infected cells and methylates VSV mRNA. The 5 VSV mRNAs are well characterized and we have developed tools necessary to define how m6Am influences the function of each of those mRNAs. Our preliminary data shows that neither mRNA stability nor mRNA translation is impacted by the loss of m6Am, and that in 293T and Hela cells in culture, virus replication is unaffected under basal conditions. Pretreatment of cells with interferon, however, demonstrates that loss of PCIF1 results in the further translational suppression of viral mRNA and a more pronounced reduction in viral growth. This PCIF1 dependent phenotype suggests that one function of this cap-proximal m6Am is to discriminate self from non-self mRNA. The mRNA cap has been hypothesized to have emerged with eukaryotic evolution, when PCIF1 is first detected, to replace the Shine-Dalgarno sequence for directing ribosomes to mRNAs and to protect mRNAs from digestion by 5' exoribonucleases thus providing an early method for distinguishing self- versus foreign mRNAs. It is likely that extant viruses have evolved in the face of this RNA methylation to evade the eukaryotic self-defense system. To further probe the role of PCIF1 modification of viral RNA we generated a PCIF1 -/- mouse providing an additional unique reagent to mechanistically dissect the role of m6Am of viral mRNA in vivo. Capitalizing on this preliminary data we will use genetic, biochemical, cell biological and virological approaches both in cell-culture and in vivo to dissect the role of m6Am and PCIF1 mediated mRNA methylation. Our underlying hypothesis is that PCIF1 modification of mRNA contributes to distinguishing self from non-self mRNA, and that viruses have coopted PCIF1 to ensure efficient replication.