Fig. 2: Transcript-specific analysis reveals adenovirus RNAs contain METTL3-dependent m6A modifications.
a The viral transcriptome is schematized with forward facing transcripts above the genome and reverse transcripts below. Viral gene kinetic classes are color-coded to denote early (gray) or late (black) genes. Lines with arrows denote introns, thin bars are untranslated exonic regions, and thick bars represent open reading frames. The names of each viral transcriptional unit are shown below the transcript cluster. meRIP-Seq was performed in triplicate on Ad5-infected A549 cells at 24 hpi. Representative meRIP data (blue/red) and total input RNA (light blue/yellow) sequence coverage is plotted against the adenovirus genome. Peaks containing increased meRIP-seq signal over input were called with MACS2 and denoted by blue boxes. Using direct RNA (dRNA) sequencing, full-length RNAs were sequenced from A549 parental cells or METTL3 knockout cells infected with adenovirus for 24 h. Specific m6A sites were predicted by comparing the nucleotide error rate of dRNA sequence data from WT to KO cells. Indicated in purple vertical lines are individual adenosines predicted to be modified by m6A that reach statistical significance when applied to all RNA that maps to a single nucleotide of the Ad genome (dRNA Exome). All Ad5-mapping transcripts were binned into unique full-length reads spanning entire transcript isoform and the same m6A prediction was applied on a transcript-by-transcript basis. Magenta vertical lines indicate predicted m6A residues found on the transcriptome level (dRNA Isoform). In addition, the position of m6A present in each viral transcript is highlighted in magenta directly on the transcript schemes. b HOMER reveals nucleotide motifs through analysis of MACS2 called peaks in cellular meRIP-seq data from Mock or Ad5-infected samples. Statistical significance was determined using hypergeometric enrichment calculations to find enriched motifs, and p-value was corrected for multiple testing. c Metagene analysis of m6A-peak distribution across cellular mRNA molecules containing 5’ and 3’ untranslated regions (UTR) and coding sequence (CDS) in Mock or Ad5-infected samples. d meRIP-qRT-PCR was performed on total RNA isolated from Ad5-infected control or METTL3 knockdown A549 cells 24 h post-infection. e Immunoblot showing knockdown efficiency of METTL3 in A549 cells. f Representative immunoblot showing two clones generated from Cas9-mediated knockout of METTL3 in A549 cells. For all assays, significance was determined by unpaired two-tailed Student’s T-test, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant. Exact p-Values are included in the source data file. Sequencing experiments are representative of three biological replicates for Illumina data and two biological replicates analyzed in a four-way comparison for Nanopore data. Immunoblots in panels e and f were independently performed at least three times. Graphs represent mean +/− standard deviation.
