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Abstract
RNA modifications have been shown to regulate all aspects of RNA fates, yet their role on chromatin-associated RNA (caRNA) has only been recently recognized. Due to the regulatory function of caRNA, RNA modifications on it could impact not only the fate of the modified RNA, but also the chromatin environment. Here, we focused on two modifications and investigated their regulatory roles on gene expression through caRNA. First, we revealed the function of RNA m5C by exploring the mechanism of 5-azacytidine (5-azaC), a first-line therapy for myeloid dysplastic syndrome and acute myeloid leukemia. Despite being traditionally characterized as a DNA hypomethylating agent, we found that 5-azaC also depletes RNA m5C, which is sufficient to kill leukemia cells with impeded cell cycle and impaired DNA damage response, causing apoptosis in the treated cells. Contrary to the canonical view, 5-azaC causes transcriptional repression, supporting its functions in cellular pathways. Mechanistically, the depleted m5C on caRNA impaired MBD6 recruitment, which further impacted PR-DUB recruitment and its H2AK119ub removal, leading to upregulation of H2AK119ub. In parallel, m5C depletion on caRNA also disrupted recruitment of SRSF2 and its interactor p300, causing downregulated H3K27ac. The RNA dependent function of 5-azaC can be recapitulated by NSUN2 depletion, supporting the causal link between RNA m5C and the cellular stress. We further identified TET2 and IKZF1, whose mutation or depletion can sensitize leukemia towards 5-azaC. Overall, we revealed a previously unrecognized role of RNA m5C in leukemia treatment by 5-azaC, providing strategies for future therapy development. In addition to m5C, we also profiled 2’-O-methylation (Nm) on caRNA, which is highly abundant in the intronic regions and regulates splicing of modified transcripts. Given the previously unrecognized nuclear function of Nm, we further explored Nm-binding proteins for the first time and identified the nuclear localized RNA-binding protein FUBP1, which is responsible for the Nm-dependent splicing regulation on caRNA. In summary, our work added new aspects of caRNA-mediated gene expression regulation by m5C and Nm.