MDM2 binds and suppresses RNA polymerase III to restrain the innate immune response to cytosolic DNA

The oncoprotein MDM2 is widely recognized as the principal negative regulator of p53, thereby controlling the expression of numerous RNA polymerase II (Pol II)-dependent genes involved in cell cycle arrest and apoptosis. Beyond this canonical role, MDM2 also engages in transcriptional regulation independently of p53, for instance through interactions with polycomb repressor complexes. Here, we identify RNA polymerase III (Pol III) as an additional target of MDM2 function. Using two complementary chemical tools – a small-molecule MDM2 antagonist (MI-1061) that releases p53 and augments MDM2 expression, and a PROTAC degrader (MD-224) that simultaneously activates p53 and promotes MDM2 ubiquitination and proteasomal destruction – we dissected the role of MDM2 in cells with amplified MDM2. Elevated MDM2 suppressed the transcription of Pol III-dependent genes encoding tRNA or 5S ribosomal RNA. Mechanistically, we found the aminoterminal domain of MDM2 associated with the catalytic subunit of Pol III, revealing a molecular link between MDM2 and the Pol III machinery. Because Pol III also acts as a cytosolic DNA sensor that converts DNA into double-stranded RNA to trigger RIG-I–TBK1–IRF3–dependent signaling, we asked whether MDM2 modulates this pathway. Indeed, enhanced MDM2 expression markedly attenuated the induction of innate immunity genes such as CXCL10, OAS1, and MX1 following transfection with Poly(dA:dT). Similarly, DNA damage induced by a radiomimetic agent activated Pol III–dependent innate signaling, and this was again blunted by high MDM2 levels, resulting in enhanced cell survival. Taken together, our findings establish MDM2 as a previously unrecognized suppressor of Pol III–dependent transcription in response to cytosolic DNA. This expands the repertoire of MDM2 functions beyond repression of p53 and Pol II activity, positioning MDM2 as a broad regulator of transcription and innate immunity.