35Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. 36Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the 37 addition of N6-methyladenosine (m 6 A) to mRNAs. While m 6 A-modified adenoviral RNAs have 38 been previously detected, the location and function of this mark within the infectious cycle is 39 unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that 40 would impede accurate m 6 A mapping using short-read sequencing, we profiled m 6 A within the 41 adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read 42 sequencing to yield both nucleotide and transcript-resolved m 6 A detection. Although both early 43and late viral transcripts contain m 6 A, depletion of m 6 A writer METTL3 specifically impacts viral 44 late transcripts by reducing their splicing efficiency. These data showcase a new technique for 45 m 6 A discovery within individual transcripts at nucleotide resolution, and highlight the role of m 6 A 46 in regulating splicing of a viral pathogen. 47
Introduction 48Adenovirus is a nuclear-replicating DNA virus with a linear double-stranded genome that 49 is dependent on the host cell machinery for productive infection 1 . To maximize coding capacity 50 of the 36 kilobase genome, adenovirus employs a tightly regulated gene transcription process. 51 Early genes and subsequent late genes are produced from both strands of DNA using cellular 52 RNA polymerase II and the spliceosomal machinery to generate capped, spliced, and 53 polyadenylated messenger RNAs (mRNA). Besides the four canonical ribose nucleosides, 54 adenoviral RNA is also known to contain RNA modifications 2,3 . RNA modifications comprise a 55 family of over one hundred chemical modifications of nucleic acid that can play important roles 56 in both RNA biogenesis and function 4,5 . In eukaryotic messenger RNA, N6-methyladenosine 57 (m 6 A) is the most prevalent RNA modification besides the 7-methylguanosine cap 6 . This mark 58 has been implicated in regulating multiple processes of RNA maturation, including splicing, 59 polyadenylation, export, translation, and ultimately decay 7-12 . Current understanding suggests 60 that m 6 A is added to messenger RNAs co-transcriptionally in the nucleus by recruitment of a 61 writer complex composed of METTL3, METTL14, WTAP, and other accessory proteins to RNA 62 polymerase II [13][14][15] . These modified mRNAs are bound by reader proteins such as the YTH 63 family (YTHDC1-2, YTHDF1-3) 16,17 , various hnRNPs 18,19 , and the IGF2B 20 family of RNA binding 64 proteins, which affect various downstream fates of the modified mRNAs. This mark is reversible 65 through the action of erasers, and FTO and ALKBH5 have been proposed to demethylate 66 m 6 A 8,21,22 . 67Shortly after the discovery of m 6 A in cellular RNAs, RNAs from several diverse viruses 68 such as adenovirus, Rous sarcoma virus, simian virus 40, herpes simplex virus, and influenza A 69 virus were also shown t...