What Is Internal m7G in RNA and What Can m7G MeRIP Sequencing Tell Us?

[Diannagellar]

The m7G modification on the Hoogsteen edge does not interfere with reverse transcription, but it is sensitive to sodium borohydride (NaBH4) reduction, which has been used to create abasic sites at m7G modified RNA positions. The RNA will be cleaved at the abasic sites after treatment with aniline, allowing reverse transcription stops to be detected by mapping. This strategy was used in conjunction with an m7G specific antibody-based method (can be found at m7G MeRIP-seq) to confidently map the m7G modifications on mouse tRNAs using the tRNA reduction and cleavage sequencing (TRAC-Seq method). Two more papers describing m7G mapping methods were published recently, while this manuscript was being reviewed. Both studies merged results from m7G MeRIPseq with results from RNA reduction with NaBH4 and biotinylation of abasic sites with biotin reagents. Pandolfini et al. created the Borohydride Reduction Sequencing (BoRed-seq) method, which is based on the pull-down of biotinylated RNA fragments followed by sequencing and was applied to human miRNAs, which resulted in the discovery of several human miRNAs with m7G modifications, involving Let-7e, which was further categorized. Zhang et al. used their methodologies to map 803 m7G mRNA modifications in human mRNA as well as m7G modifications in human tRNAs, detecting m7G modifications at nucleotide resolution as a result of variations occurring as a result of misincorporation at the biotinylated sites during reverse transcription.