RNA m⁵C modification upregulates E2F1 expression in a manner dependent on YBX1 phase separation and promotes tumor...

5-Methylcytosine (m⁵C) is a common RNA modification that modulates gene expression at the posttranscriptional level, but the crosstalk between m⁵C RNA modification and biomolecule condensation, as well as transcription factor-mediated transcriptional regulation, in ovarian cancer, is poorly understood. In this study, we revealed that the RNA methyltransferase NSUN2 facilitates mRNA m⁵C modification and forms a positive feedback regulatory loop with the transcription factor E2F1 in ovarian cancer. Specifically, NSUN2 promotes m⁵C modification of E2F1 mRNA and increases its stability, and E2F1 binds to the NSUN2 promoter, subsequently reciprocally activating NSUN2 transcription. The RNA binding protein YBX1 functions as the m⁵C reader and is involved in NSUN2-mediated E2F1 regulation. m⁵C modification promotes YBX1 phase separation, which upregulates E2F1 expression. In ovarian cancer, NSUN2 and YBX1 are amplified and upregulated, and higher expression of NSUN2 and YBX1 predicts a worse prognosis for ovarian cancer patients. Moreover, E2F1 transcriptionally regulates the expression of the oncogenes MYBL2 and RAD54L, driving ovarian cancer progression. Thus, our study delineates a NSUN2-E2F1-NSUN2 loop regulated by m⁵C modification in a manner dependent on YBX1 phase separation, and this previously unidentified pathway could be a promising target for ovarian cancer treatment.