Direct RNA sequencing reveals mA modifications on adenovirus RNA are necessary for efficient splicing.

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (mA) to mRNAs. While mA-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate mA mapping using short-read sequencing, here we profile mA within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved mA detection. Although both early and late viral transcripts contain mA, depletion of mA writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for mA discovery within individual transcripts at nucleotide resolution, and highlight the role of mA in regulating splicing of a viral pathogen.