show Abstracthide AbstracttRNAs 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. Overall design: 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.