Detection of internal m7G modification in the human transcriptome in a quantitative manner and at single-nucleotide resolution

RNA modifications play an important role in regulating RNA stability and gene expression, however the development of reliable detection techniques has been a major burden in the field. Here, we describe two different methodologies for global identification of N-7-methylguanosine (m7G) in human tRNAs. An antibody and northdot blot based quantitative approach that allows global detection of m7G levels. And borohydride reduction sequencing (Bo-Seq), a chemically based approach that maps m7G modification at single nucleotide resolution. The specificity of Bo-Seq lies on RNA size-selection, optimized RNA scission using NaBH4, aniline and m7GTPs, and elimination of complex chemical steps or bioinformatic analysis included in similar methods, and reduces the protocol to four days. Bo-Seq and our antibody based approach are specifically developed for easily and globally detect tRNA m7G-methylation in human cells. We also validated METTL1 as the methyltransferase that methylates tRNAs in human cells. Small RNA from prostate cancer cells DU145 were extrated. RNA were either non chemically treated or treated wih NaBH4 and aniline (BH4). Two replicates were used per condition. A total of 4 samples were sequenced.