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.