Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing

Methylation of guanosine on position N7 (m(7)G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m(7)G Mutational Profiling sequencing (m(7)G-MaP-seq), which allows high throughput detection of m(7)G modifications at nucleotide resolution. In our method, m(7)G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m(7)G-MaP-seq efficiently detects known m(7)G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m(7)G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m(7)G modification occurs before tRNA splicing. We do not find any evidence for internal m(7)G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m(7)G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m(7)G modifications.