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

细胞甲基转移酶 PCIF1 对病毒 mRNA 进行帽特异性 N6 甲基化

• 批准号: 10456693
• 负责人: Eric Lieberman Greer
• 金额: $ 75.26万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-26 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10456693
• 关键词: 5' -exoribonuclease; Adenosine; Affect; Animals; Antiviral Response; Attenuated; Binding; Biochemical; Biochemical Genetics; Biological; C-terminal; Cell Culture Techniques; Cell Nucleus; Cells; Cytoplasm; Data; Digestion; Discrimination; Elements; Embryo; Ensure; Evolution; Fibroblasts; Genes; Genetic; Genetic Translation; Genomic approach; Goals; Growth; Guanine; Hela Cells; In Vitro; Infection; Interferon-beta; Interferons; Knock-out; Literature; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; Modification; Mus; Nuclear Export; Phenotype; Positioning Attribute; Publishing; RNA; RNA Polymerase II; RNA Sequences; RNA Viruses; RNA methylation; Reagent; Ribose; Ribosomes; Role; Structure; System; Testing; Transcript; Transcription Initiation Site; Translations; Vesicular stomatitis Indiana virus; Vesicular stomatitis virus M protein; Viral; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; epitranscriptomics; in vivo; mRNA Export; mRNA Stability; mRNA capping; mutant; tool; transcription factor TFIIH; transcriptome; viral RNA

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.

该项目的长期目标是确定存在于真核 mRNA 转录起始位点的帽子特异性 N6, 2'-O-二甲基腺苷 (m6 Am) 的功能。我们和其他人最近将负责 N6 位置近端腺苷甲基化的细胞 mRNA 甲基转移酶鉴定为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 端结构域，选择性修饰帽近端 A，随后鸟嘌呤-N7-甲基化酶和核糖-2'-O 甲基化酶对帽结构进行连续甲基化。帽近端 m6Am 修饰的功能意义尚不确定，已发表的文献对 mRNA 稳定性和翻译得出了不同的结论。在细胞质中复制的病毒，例如负链 RNA 病毒水泡性口炎病毒 (VSV)，尽管不存在病毒编码的 N6, 2'-O-二甲基转移酶，但在受感染细胞中合成的 mRNA 上也含有这种近端 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，并使 VSV mRNA 甲基化。 5 个 VSV mRNA 已得到很好的表征，我们开发了必要的工具来定义 m6Am 如何影响每个 mRNA 的功能。我们的初步数据表明，mRNA 稳定性和 mRNA 翻译均不受 m6Am 丢失的影响，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，用干扰素预处理细胞表明，PCIF1 的缺失会导致病毒 mRNA 的进一步翻译抑制和病毒生长的更明显减少。这种 PCIF1 依赖性表型表明这种近端 m6Am 的功能之一是区分自身 mRNA 和非自身 mRNA。据推测，当首次检测到 PCIF1 时，mRNA 帽随着真核进化而出现，以取代 Shine-Dalgarno 序列，将核糖体引导至 mRNA，并保护 mRNA 免遭 5' 核糖核酸外切酶消化，从而提供区分自身 mRNA 与外源 mRNA 的早期方法。现存的病毒很可能是在这种RNA甲基化的情况下进化而来的，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，我们制备了 PCIF1 -/- 小鼠，提供了额外的独特试剂来机械剖析病毒 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、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 mRNA 的修饰有助于区分自身和非自身 mRNA，并且病毒利用 PCIF1 来确保有效复制。