THRONE: A New Approach for Accurate Prediction of Human RNA N7-Methylguanosine Sites

Summary 7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.

tRNAs are subject to numerous modifications including methylation. Mutations in the human N 7 -methylguanosine (m 7 G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation yet the molecular and cellular function in mammals is not well understood. We developed m 7 G methylated tRNA immunoprecipitation sequencing (MeRIP-Seq) and tRNA reduction and cleavage sequencing (TRAC-Seq) to reveal the m 7 G 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 m 7 G tRNA methylome and highlights its essential role in ESCs with links to human disease. Lin and Liu et al. developed two independent methods, MeRIP-Seq and TRAC-Seq, to profile the m 7 G tRNA methylome in mouse ESCs and revealed that Mettl1/Wdr4- mediated m 7 G tRNA methylome is required for normal mRNA translation and ESC self- renewal and differentiation.

N 7 -methylguanosine (m 7 G) is a positively-charged, essential modification at the 5’ cap of eukaryotic messenger RNA (mRNA), regulating mRNA export, translation, and splicing. m 7 G also occurs internally within transfer RNA (tRNA) and ribosomal RNA (rRNA), but its existence and distribution within eukaryotic mRNA remains to be investigated. Here, we show the presence of internal m 7 G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m 7 G methylome using m 7 G -MeRIP-seq. To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m 7 G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m 7 G-seq enabled transcriptome-wide mapping of m 7 G in human tRNA and mRNA, revealing distribution features of the internal m 7 G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m 7 G within mRNA and showed that internal m 7 G methylation could affect mRNA translation. Zhang et al. discovered the presence of internal N 7 -methylguanosine (m 7 G) within mammalian mRNA. Both antibody-based and chemical-assisted methods were developed for transcriptome-wide mapping of internal m 7 G, with the latter reaching single-base resolution. METTL1/WDR4 was identified as a writer complex that installs a subset of m 7 G on mRNA, which affects translation.