The m7G tRNA methylome regulates embryonic stem cell self-renewal and differentiation

tRNAs are subject to numerous modifications including methylation. Mutations in the human N7-methylguanosine (m7G) methyltransferase complex METTL1-WDR4 cause primordial dwarfism and brain malformation yet the molecular and cellular function in mammals is not well understood. We developed m7G methylated tRNA immunoprecipitation sequencing (MeRIP-Seq) and tRNA reduction and cleavage sequencing (TRAC-Seq) to reveal the m7G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs are modified at a ‘RAGGU’ motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m7G tRNA methylome and highlights its essential role in ESCs with links to human disease. tRNA m7G MeRIP-Seq and TRAC-Seq were developed to identify the tRNA m7G methylome in mouse embryonic stem cells. RNA-seq was used to study the differential gene expression in the Mettl1 knockout and control cells. Riboseq was used to study the differentially translated genes in the Mettl1 knockout and control cells.

转运RNA（transfer RNA, tRNA）会经历包括甲基化在内的多种修饰。人类N7-甲基鸟苷（m7G）甲基转移酶复合物METTL1-WDR4发生突变可引发原发性侏儒症与脑部畸形，但哺乳动物中该复合物的分子与细胞功能尚未得到充分阐明。我们开发了m7G甲基化tRNA免疫沉淀测序（methylated tRNA immunoprecipitation sequencing, MeRIP-Seq）与tRNA还原切割测序（tRNA reduction and cleavage sequencing, TRAC-Seq），以解析小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）中的m7G tRNA甲基化组。本研究鉴定出22种tRNA在其可变环内的‘RAGGU’基序处发生修饰。我们观察到，在Mettl1敲除的小鼠胚胎干细胞中，对应密码子的核糖体占据量显著升高，提示该修饰对tRNA功能、核糖体暂停及mRNA翻译存在广泛影响。细胞周期相关基因以及与脑部畸形相关基因的翻译尤其易受影响。Mettl1或Wdr4敲除的小鼠胚胎干细胞会出现自我更新缺陷与神经分化异常。本研究揭示了哺乳动物m7G tRNA甲基化组的复杂性，并明确了其在胚胎干细胞中的关键作用，且该作用与人类疾病密切相关。本研究开发的tRNA m7G MeRIP-Seq与TRAC-Seq技术可用于鉴定小鼠胚胎干细胞中的tRNA m7G甲基化组；通过RNA测序（RNA-seq）分析Mettl1敲除组与对照组细胞的差异基因表达；利用核糖体测序（Ribo-seq）分析两组细胞的差异翻译基因。