IntroductionThe tumor suppressor p53 is a central regulator of cellular stress responses and is frequently altered in human cancer. Mutations in the TP53 gene occur in approximately 50% of all malignancies, underscoring its critical role in maintaining genomic integrity and preventing malignant transformation [1, 2]. In its wild-type form, p53 functions as a transcription factor that induces a wide range of genes involved in cell cycle arrest, DNA repair, senescence, or apoptosis. For instance, transcriptional activation of CDKN1A/p21 enforces a G1/S checkpoint and halts proliferation, while induction of pro-apoptotic genes such as BAX, PUMA/BBC3, and NOXA/PMAIP1 initiates programmed cell death when damage is irreparable. In addition, p53 upregulates the expression of MDM2, which in turn represses p53 activity, thereby establishing a negative feedback loop that tightly controls the magnitude and duration of the p53 response.MDM2 is thus well established as the principal antagonist of p53. As an E3 ubiquitin ligase of the RING family, MDM2 not only binds to the aminoterminal transactivation domain of p53 and blocks its transcriptional activity, but also promotes its ubiquitination and proteasomal degradation. This dual mode of inhibition ensures that unchecked p53 activation does not lead to unnecessary or excessive cell cycle arrest and apoptosis [3]. Importantly, amplification of MDM2 or dysregulation of its activity is a common oncogenic event, effectively suppressing residual wild-type p53 function in many cancers, especially liposarcoma and osteosarcoma [4].Beyond its canonical role in p53 inhibition, MDM2 exerts a range of p53-independent functions, particularly in the regulation of transcription and genome stability [5]. For example, we have previously shown that MDM2 associates with polycomb repressor complexes 1 and 2, thereby modulating chromatin structure and transcriptional repression independently of p53 [6, 7]. Furthermore, MDM2 enhances the progression of DNA replication forks by preventing the accumulation of DNA:RNA hybrids, also known as R-loops [5, 8, 9]. Moreover, the interaction of MDM2 with PARP1 supports replication fork restart following stalling [10], thereby fostering S phase progression despite replicative stress. Collectively, these findings suggest that MDM2 serves as a versatile regulator of gene expression and DNA synthesis, extending well beyond its traditional function as a p53 antagonist.Up to now, however, the transcriptional role of MDM2 has been studied almost exclusively in the context of RNA polymerase II (Pol II), the enzyme responsible for pre-mRNA synthesis. Yet, eukaryotic cells contain additional distinct RNA polymerases, each with specialized roles in transcription. Among these, RNA polymerase III (Pol III) occupies a central position by generating essential structural RNAs. Pol III is a multi-subunit enzyme complex [11,12,13,14] that synthesizes transfer RNAs (tRNAs), the 5S ribosomal RNA (5S rRNA), several small nuclear and cytoplasmic RNAs such as U6 and U6atac spliceosomal RNAs, 7SK and 7SL RNAs, Y RNA, RNase P RNA, and Vault RNA [15], as well as other classes of ncRNA [16]. Thus, Pol III activity is tightly linked to cell growth and proliferation, and its dysregulation has been implicated in oncogenesis and other diseases [17].In addition to its role in housekeeping transcription, Pol III has emerged as an important mediator of innate immunity. This role is not confined to the nucleus, in agreement with the assembly of Pol III in the cytosol [18]. Cytosolic DNA, which arises during viral infection or from damaged chromosomes, can activate Pol III–dependent signaling. DNA fragments may persist in the cytosol after double-strand DNA breaks followed by mitosis, but retrotransposons or damaged mitochondria can also represent sources of cytosolic DNA [19]. While cytosolic DNA is often recognized by the cGAS–STING pathway [20, 21], Pol III provides an additional layer of detection: it transcribes cytosolic DNA in both directions, generating double-stranded RNA products with a 5′-triphosphate moiety [22,23,24,25]. These RNAs mimic viral replication intermediates and are sensed by the RIG-I–MAVS pathway [26, 27]. Specifically, RIG-I and related receptors recognize the RNA products and associate with MAVS on the outer mitochondrial membrane, which in turn activates the kinase TBK1 and the transcription factor IRF3 [28]. Phosphorylated IRF3 dimerizes and translocates to the nucleus, driving the transcription of antiviral and immunomodulatory genes, including chemokine-encoding genes such as CXCL10 and interferon-stimulated genes like OAS1 and MX1 [29]. This cascade provides a robust defense mechanism against DNA viruses and contributes to the cellular response to endogenous DNA damage.Despite the recognized role of MDM2 in modulating Pol II–dependent transcription, it has remained unclear whether MDM2 might also influence other transcriptional machineries such as Pol III, and by extension, whether MDM2 impacts the innate immune pathways that rely on Pol III activity. Given that MDM2 amplification is a frequent oncogenic event, such a mechanism could provide a means for cancer cells to suppress immune detection and evade cell death triggered by cytosolic DNA signaling.Here we report that MDM2 directly interacts with RNA polymerase III and suppresses its transcriptional activity. Using an MDM2 antagonist (MI-1061), which disrupts p53 binding and elevates MDM2 expression [30], in comparison with a proteolysis targeting chimera (PROTAC) degrader (MD-224), which additionally promotes MDM2 ubiquitination and proteasomal destruction [31, 32], we show that MDM2 levels inversely correlate with the transcription of Pol III products, including tRNA and 5S rRNA. Mechanistically, we demonstrate that the aminoterminal domain of MDM2 binds to the Pol III catalytic subunit. Functionally, MDM2 overexpression attenuates Pol III–mediated innate immune signaling, blunting RIG-I–TBK1–IRF3 activation and reducing the induction of CXCL10, OAS1, and MX1 in response to cytosolic DNA or DNA damage. By dampening Pol III activity, MDM2 suppresses innate immune responses and promotes cell survival under genotoxic stress. These findings expand the role of the MDM2 oncoprotein from a p53/Pol II regulator to a broader modulator of transcription and innate immunity.ResultsElevated MDM2 levels suppress the expression of Pol III–transcribed genesTo investigate whether MDM2 modulates RNA polymerase III (Pol III) activity, we manipulated cellular MDM2 levels using two previously described small molecules with matched scaffolds [30,31,32]. MI-1061 is a Nutlin-like compound that binds the p53-interacting pocket of MDM2, thereby displacing p53 and allowing p53 to activate transcription of its target genes. MD-224 contains the same MDM2-binding moiety that inhibits p53 interaction but is additionally linked to a Cereblon-binding group, converting it into a PROTAC that promotes MDM2 ubiquitination and degradation (Fig. 1A). Recent literature suggests that MD-224 can also destabilize the nuclear receptor PXR [33], but the gene encoding PXR is not detectably expressed in any of the cell lines under study here, according to proteinatlas.org [34].Fig. 1: MDM2 accumulation inhibits RNA Pol III activity.Full size imageA Schematic representation of the proteolysis targeting chimera (PROTAC) MD-224 used in this study. The MDM2-binding moiety MI-1061 is linked to a ligand of the E3 ligase Cereblon to form a PROTAC, which mediates MDM2 ubiquitination and subsequent proteasomal degradation. B Immunoblot analysis of whole-cell lysates from SJSA-1 cells treated with the MDM2-binding antagonists Nutlin (20 μΜ) or MI-1061 (1 μM), or with the PROTAC MD-224 (1 μM), for 12 h, reflecting the accumulation of p53 and its target gene products upon treatment with MDM2 antagonists. Of note, the accumulation of MDM2 was reduced in the presence of the PROTAC MD-224. Replicates and quantification of the protein levels are shown in Supplementary Fig. 1A. C Expression of the p53 target genes MDM2 and CDKN1A in SJSA-1 cells treated as in (B). Transcript levels were quantified by RT-qPCR, normalized to 36B4 mRNA and depicted relative to the control; n = 4. D Top: Metabolic pulse-labeling of nascent RNAs at multiple time points following the addition of Nutlin (20 µM), MI-1061 (1 µM) or MD-244 (1 µM). Bottom: Quantification of nascent 5S rRNA and tRNA levels shown as mean ± SEM. a.u., arbitrary units; n = 3. Replicates are shown in Supplementary Fig. 1B. E Expression of RNA Pol III target genes in SJSA-1 cells. Levels of total and non-spliced tRNAs for Leu and Tyr were quantified by RT-qPCR upon treatments as in (B) and shown as means of five independent replicates ± SEM. Supplementary Fig. 1C presents the sequences of tRNAs and corresponding primers. F H1299 cells were co-transfected with expression plasmids for MDM2 and/or GFP at a 9:1 ratio for 36 h. GFP-positive cells were subsequently collected by fluorescence-activated cell sorting (FACS) and subjected to RT–qPCR analysis as in panel (E). Graphs are shown as means of three independent replicates ± SEM. For primer sequences, cf. Supplementary Fig. 1C. G Reduction in nascent 5S rRNA and tRNA levels in response to MDM2 overexpression. H1299 cells were co-transfected to express MDM2 and/or GFP, followed by FACS as in (F). GFP-positive cells were re-seeded and subjected to metabolic pulse-labeling of nascent RNAs. Quantification of the signals corresponding to 5S rRNA and tRNA is shown as means of three independent replicates ± SEM. H Volcano plots showing differential expression of nuclear encoded tRNAs, as determined by deep sequencing. SJSA-1 cells were treated with MI-1061, MD-224, or Nutlin, in comparison to the DMSO control as in (B). The plots reflect log2(fold change) vs –log10(p-value) for all quantified tRNAs. Differentially expressed tRNAs (p-value