m5C RNA modification upregulates E2F1 expression dependently on YBX1 phase separation and promotes tumor progression in ovarian cancer

Abstract 5-methylcytosine (m 5 C) is a common RNA modification that modulates gene expression at the post-transcriptional level, but the cross-talk between m 5 C RNA modification and biomolecule condensation as well as transcription factor-mediated transcriptional regulation in ovarian cancer remains poorly understood. In this study, we uncover that the RNA methytransferase NSUN2 facilitates m 5 C modification of mRNA and forms a positive feedback regulatory loop with the transcription factor E2F1 in ovarian cancer. Specifically, NSUN2 promotes m 5 C modification of E2F1 mRNA and enhances its stability, and E2F1 binds to NSUN2 promoter followed by the activated transcription reciprocally. The RNA binding protein YBX1 acts as the m 5 C reader and is involved in NSUN2-mediated E2F1 regulation. m 5 C modification promotes YBX1 phase separation that upregulates E2F1 expression. In ovarian cancer, NSUN2 and YBX1 are amplified and upregulated, and higher expressions of NSUN2 and YBX1 predict a worse prognosis for ovarian cancer patients. Moreover, E2F1 transcriptionally regulates the expression of oncogenes MYBL2 and RAD54L, driving ovarian cancer progression. Thus, our study delineates a NSUN2-E2F1-NSUN2 circuitry regulated by m 5 C modification dependently on YBX1 phase separation, and the identified previously unknown pathway can be a promising target for ovarian cancer treatment.