The bladder cancer m6A landscape is defined by global methylation dilution and focal 3'-UTR hypermethylation.

N6-Methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNAs and regulates target transcripts throughout the mRNA life cycle. Although changes in m6A have been reported in human cancers, technical limitations have hindered a comprehensive understanding of the cancer-associated m6A landscape. Here, we use GLORI-sequencing to establish the first transcriptome-wide, single-nucleotide resolution maps of m6A in bladder cancer. Comparing bladder cancer and healthy bladder samples, we discover two key m6A signatures: a global dilution of methylation and a focal hypermethylation at 3'-UTRs. The global methylation dilution results from an increased expression of unmethylated transcripts and a decreased expression of methylated transcripts. In contrast, focal 3'-UTR hypermethylation is associated with the overexpression of VIRMA, a component of the m6A writer complex. A functional role of VIRMA is confirmed in knockdown experiments that reveal reduced 3'-UTR methylation and oncogenic phenotypes of bladder cancer cells. Our study is the first to describe the m6A epitranscriptomic landscape of cancer at single-base resolution and provides first insights into the processes that generate its characteristic signatures.