Exploring the m<sup>5</sup>C epitranscriptome of mRNAs in breast cancer cells through genome engineering and long-read sequencing approaches

Epitranscriptomics has emerged as a rapidly evolving field that focused on studying post-transcriptional RNA modifications and their role in spatiotemporal regulation of gene expression. N6-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C) represent the most extensively studied modifications on mRNAs. These reversible modifications, mediated by 'writer,' 'eraser,' and 'reader' proteins, dynamically fine-tune mRNA stability, splicing, and translation. Growing evidence links their dysregulation to pathological states, including cancer progression and metastasis, where their aberrant deposition on oncogenes or tumor suppressors alters cellular signaling and therapeutic responses. In the current study, we present a detailed analysis of the m⁵C epitranscriptomic landscape across distinct breast cancer molecular subtypes. Using CRISPR/Cas9, we confirm NSUN2 as a key m⁵C writer in human mRNAs. NSUN2 loss was validated by targeted sequencing and Western blotting. Furthermore, we demonstrate the regulatory effects of NSUN2 on its canonical mRNA targets, revealing its critical role in maintaining proper gene expression networks. Our findings strongly suggest that additional m⁵C writers contribute to m⁵C methylation machinery. Additionally, we assessed the functional impact of NSUN2 depletion on mRNAs harboring m⁵C sites using mRNA stability assays. Furthermore, our analysis revealed distinct m⁵C methylation patterns among breast cancer subtypes, highlighting unique m⁵C signatures associated with the disease. Notably, we identified specific hypomethylated and hypermethylated m⁵C sites in each breast cancer cell line, representing a universal m⁵C methylation signature for breast cancer. Our study constitutes the first comprehensive m⁵C epitranscriptomic atlas in human breast cancer and paves the way for future research aimed at developing targeted therapeutic interventions that leverage the m⁵C methylation landscape.