RNF20 promotes the progression of diffuse large B-cell lymphoma through regulating ubiquitination-dependent Phase separation of fus Free

RNF20 is an E3 ubiquitin ligase known to regulate transcription, DNA repair, and chromatin architecture. Emerging evidence has found that RNF20 is involved in regulating biomolecular condensation through liquid-liquid phase separation (LLPS). LLPS enables cells to organize intracellular components into dynamic, membrane-less compartments that influence gene expression, stress response, and signal transduction. This study aims to explore the regulatory function and underlying mechanism of RNF20 in diffuse large B-cell lymphoma (DLBCL).

We first detected the expression of RNF20 in DLBCL tissues and reactive hyperplasia cases with informed consent. DLBCL tissues exhibited higher levels of RNF20 compared to the control cases. Survival analysis revealed that DLBCL patients with elevated RNF20 levels showed worse clinical prognosis.

The above findings prompted us to investigate the biological function of RNF20 in DLBCL. RNF20 knockdown declined cell proliferation, induced cell apoptosis and cell cycle arrest in the G2/M phase. Conversely, overexpression of RNF20 facilitated cell proliferation. Moreover, the oncogenic role of RNF20 was confirmed in DLBCL xenograft models. Furthermore, combined treatment of RNF20 deficiency and ibrutinib, a Bruton's tyrosine kinase inhibitor, significantly declined DLBCL cell proliferation compared to the control group.

To explore the underlying mechanism of RNF20 in DLBCL progression, we performed mass spectrometry to identify interacting proteins of RNF20. Integrating molecular docking analysis, FUS was identified as the key RNF20-interacting protein in DLBCL. Further confocal immunofluorescence and co-immunoprecipitation confirmed the colocalization and interaction between RNF20 and FUS in DLBCL cells. Moreover, FUS expression showed a significant positive correlation with RNF20, and RNF20 knockdown significantly reduced the protein expression levels of FUS in DLBCL cells. Conversely, overexpression of RNF20 facilitated the protein expression of FUS. Notably, domain truncation revealed that the interaction between RNF20 and FUS occurred based on the CC1 domain of RNF20 and the RRM domain of FUS.

We further elucidated the molecular mechanism by which RNF20 regulated the expression of FUS. Cycloheximide chase experiment showed that RNF20 knockdown decreased the half-life of FUS protein in DLBCL cells. MG132 treatment reversed this effect, indicating proteasome involvement. Importantly, RNF20 overexpression significantly increased the ubiquitination levels of FUS, which was predominantly via K63-linked chains. Next, we found that the RING domain truncation of RNF20 protein could reverse the positive role of RNF20 on FUS ubiquitination. These results suggested that RNF20 specifically mediated a non-proteolytic ubiquitination of FUS based on RING domain in DLBCL cells. Furthermore, we purified recombinant FUS and confirmed its phase separation capacity in vitro, with dynamic recovery upon fluorescence recovery after photobleaching (FRAP). Moreover, FUS overexpression promoted the formation of LLPS condensates in DLBCL cells, which also exhibited FRAP recovery. Strikingly, RNF20 overexpression enhanced the phase separation of FUS protein in both fixed and live-cell imaging, whereas RNF20 knockdown significantly reduced it. Furthermore, both the RING domain truncation of RNF20 and the mutation at the ubiquitylation sites of FUS could inhibit the phase separation of FUS in DLBCL cells, indicating the key role of RNF20 in ubiquitination-dependent phase separation of FUS. Importantly, overexpression of FUS could partially reverse the anti-tumor effects of targeting RNF20 in DLBCL. To decipher the specific contribution of RNF20-FUS axis in DLBCL pathogenesis, FUS ChIP-seq was employed, and GO analysis revealed that after RNF20 overexpression, FUS preferentially activated genes involved in redox regulation, metabolic processes, and NADH dehydrogenase activity, which are critical for cellular energy metabolism and survival. Taken together, these findings suggested that RNF20 mediated FUS ubiquitination, affecting its protein stability and ultimately promoting FUS phase separation and transactivation of downstream genes in DLBCL.

In conclusion, our results revealed that RNF20 induced the ubiquitination-dependent phase separation of FUS, which promoted the malignant progression of DLBCL. This underscores the potential of RNF20 as a novel prognostic biomarker and therapeutic target for DLBCL.