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

该项目的长期目标是确定真核mrna转录起始位点的帽特异性n6,2 '- o -二甲基腺苷（m6 Am）的功能。我们和其他人最近发现，负责N6位置近端腺苷甲基化的细胞mRNA甲基转移酶是磷酸化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，在鸟嘌呤- n7甲基化酶和核糖-2'- o甲基化酶的顺序甲基化之后，选择性地修饰帽近端A。帽端-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），尽管缺乏病毒编码的n6,2 '- o -二甲基转移酶，但在感染细胞中合成的mRNA上也含有这种帽端-近端m6Am修饰。在初步数据中，我们发现PCIF1重新定位到VSV感染细胞的细胞质中，并甲基化VSV mRNA。5种VSV mrna已经被很好地表征，我们已经开发了必要的工具来定义m6Am如何影响每种mrna的功能。我们的初步数据显示，mRNA的稳定性和mRNA的翻译都不受m6Am缺失的影响，并且在培养的293T和Hela细胞中，病毒复制在基础条件下不受影响。然而，用干扰素对细胞进行预处理表明，PCIF1的缺失导致病毒mRNA的进一步翻译抑制和更明显的病毒生长减少。这种PCIF1依赖性表型表明，帽端m6Am的一个功能是区分自我和非自我mRNA。mRNA帽被认为是在真核生物进化过程中出现的，当PCIF1首次被检测到时，它取代了Shine-Dalgarno序列，将核糖体引导到mRNA上，并保护mRNA不被5'外核糖核酸酶消化，从而提供了一种区分自体和外源mRNA的早期方法。现存的病毒很可能在面对这种RNA甲基化的情况下进化，以逃避真核生物的自卫系统。为了进一步探索PCIF1修饰病毒RNA的作用，我们构建了PCIF1 -/-小鼠，提供了一种额外的独特试剂来机械地解剖体内病毒mRNA的m6Am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、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修饰mRNA有助于区分自我和非自我mRNA，病毒采用PCIF1来确保有效复制。

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.