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. Overall design: 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.