Direct RNA sequencing reveals m⁶A 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 (m⁶A) to mRNAs. While m⁶A-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 m⁶A mapping using short-read sequencing, here we profile m⁶A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m⁶A detection. Although both early and late viral transcripts contain m⁶A, depletion of m⁶A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m⁶A discovery within individual transcripts at nucleotide resolution, and highlight the role of m⁶A in regulating splicing of a viral pathogen.