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Over 150 types of RNA modifications are identified in RNA molecules. Transcriptome profiling is one of the key steps in decoding the epitranscriptomic panorama of these chemical modifications and their potential functions. N 7 -methylguanosine (m 7 G) is one of the most abundant modifications present in tRNA, rRNA and mRNA 5'cap, and has critical roles in regulating RNA processing, metabolism and function. Besides its presence at the cap position in mRNAs, m 7 G is also identified in internal mRNA regions. However, its transcriptome-wide distribution and dynamic regulation within internal mRNA regions remain unknown. Here, we have established m 7 G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (m 7 G miCLIP-seq) to specifically detect internal mRNA m 7 G modification. Using this approach, we revealed that m 7 G is enriched at the 5'UTR region and AG-rich contexts, a feature that is well-conserved across different human/mouse cell lines and mouse tissues. Strikingly, the internal m 7 G modification is dynamically regulated under both H 2 O 2 and heat shock treatments, with remarkable accumulations in the CDS and 3'UTR regions, and functions in promoting mRNA translation efficiency. Consistently, a PCNA 3'UTR minigene reporter harboring the native m 7 G modification site displays both enriched m 7 G modification and increased mRNA translation upon H 2 O 2 treatment compared to the m 7 G site-mutated minigene reporter (G to A). Taken together, our findings unravel the dynamic profiles of internal mRNA m 7 G methylome and highlight m 7 G as a novel epitranscriptomic marker with regulatory roles in translation.

Dynamic methylome of internal mRNA N7-methylguanosine and its regulatory role in translation