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

本项目的长期目标是确定存在于真核生物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。我们和其他人最近确定了细胞mRNA甲基转移酶负责甲基化的帽近端腺苷在N6位置作为磷酸化的羧基末端结构域相互作用因子1（PCIF 1）。PCIF 1结合宿主RNA聚合酶II的磷酸化C末端结构域，以选择性地修饰帽近端A，随后通过鸟嘌呤-N7-甲基化酶和核糖-2 '-O甲基化酶顺序甲基化帽结构。帽近端m6 Am修饰的功能意义尚不确定，已发表的文献对mRNA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）也在感染细胞中合成的mRNA上含有这种帽近端m6 Am修饰，尽管不存在病毒编码的N6，2 '-O-二甲基转移酶。在初步数据中，我们发现PCIF 1在VSV感染的细胞中重新定位于细胞质并使VSV mRNA甲基化。5个VSV mRNA被很好地表征，并且我们已经开发了必要的工具来定义m6 Am如何影响这些mRNA中的每一个的功能。我们的初步数据显示，mRNA稳定性和mRNA翻译都不受m6 Am丢失的影响，并且在培养的293 T和Hela细胞中，病毒复制在基础条件下不受影响。然而，用干扰素预处理细胞表明，PCIF 1的缺失导致病毒mRNA的进一步翻译抑制和病毒生长的更明显减少。这种PCIF 1依赖性表型表明，这种帽近端m6 Am的一个功能是区分自身与非自身mRNA。已经假设mRNA帽在首次检测到PCIF 1时随着真核生物进化而出现，以取代Shine-Dalgarno序列用于将核糖体引导至mRNA并保护mRNA免受5'核糖核酸外切酶的消化，从而提供区分自身mRNA与外源mRNA的早期方法。现存的病毒很可能在面对这种RNA甲基化时进化，以逃避真核生物的自我防御系统。为了进一步探索病毒RNA的PCIF 1修饰的作用，我们产生了PCIF 1-/-小鼠，提供了另外的独特试剂来机械地剖析病毒mRNA的m6 Am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、生物化学、细胞生物学和病毒学方法来剖析m6 Am和PCIF 1介导的mRNA甲基化的作用。我们的基本假设是，PCIF 1修饰的mRNA有助于区分自我和非自我的mRNA，以及病毒已增选PCIF 1，以确保有效的复制。

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.