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 used GLORI-sequencing to establish the first transcriptome-wide, single-nucleotide resolution maps of m6A in cancer. Differentially methylated transcripts were enriched in oncogenic pathways relevant to urothelial carcinoma of the bladder (UCB). We discovered two key m6A signatures in UCB: a global loss of methylation and a local hypermethylation at 3'-UTRs. Integration of RNA-sequencing data revealed that the global loss resulted from dilution of methylation marks due to increased expression of unmethylated transcripts and decreased expression of highly methylated transcripts. In contrast, local 3'-UTR hypermethylation was associated with the overexpression of VIRMA, a component of the m6A writer complex, which was linked to UCB progression. 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. Overall design: Using RNA sequencing, we analyzed transcript expression changes in nine tumoral and nine paratumoral patient samples. Furthermore, we investigated alternative polyadenylation events in the same patient samples and in the UCB cell lines T24 and UM-UC-3, in which we knocked out the m6A methyltransferase METTL3. Also, the cells were treated with DMSO (control) and with 50 µM of the METTL3 inhibitor STM2457. Three biological replicates were sequenced per condition and cell line. Additionally, we sequenced three biological replicates of RT4/UM-UC-3 shCtrl and shVIRMA cells for investigating the role of VIRMA in alternative polyadenylation in UCB.