Nono Recruits Ribosomal Protein RPLP0 to DNA Damage Sites to Enhance Non-Homologous End Joining and Tumor Radio-Resistance

Abstract

Colorectal cancer (CRC) is one of the most common and deadly cancers in the world. Currently, radio-therapy is one of the major treatments for CRC. NONO, an RNA/DNA binding protein, has been known to enhance radio-resistance by accelerating DNA repair, whereas its underlying mechanism remains largely unknown. In this study, we aimed to explore its molecular mechanism and clinical significance in CRC. The radiation-induced NONO-binding protein was identified by Co-immunoprecipitation (CoIP) and mass spectrum (MS). The association between proteins was verified by CoIP, immunofluorescence and proximal ligation assay. The expression of RNAs and proteins were evaluated by quantitative real-time PCR and western blotting, respectively. The DNA damage level was evaluated by the analyses of γ-H2A.X level and counting of γ-H2A.X foci. The double strand break repair efficiency was examined by in vitro non-homologous end joining (NHEJ) assay. NONO knockout enhanced radio-sensitivity, reduced the repair of radiation-induced DNA damage and NHEJ efficiency. Through CoIP and MS, RPLP0 was identified as a novel NONO-binding protein, and their association was further enhanced upon irradiation. NONO bound to free RPLP0, but not that within ribosome, in nucleus. The RPLP0-binding domain of NONO was mapped to RRM1/2 domain. Furthermore, RPLP0 silencing reduced the repair of radiation-induced DNA damage and the efficiency of NHEJ. As translation blockage with Cycloheximide had no influence on DNA damage repair, RPLP0 silencing-induced DNA repair deficiency was not a result of translation inhibition. Mechanically, RPLP0 was recruited to DNA damage site by NONO and enhanced DNA repair. The analyses of clinical samples showed that both the RNA and protein level of NONO and RPLP0 were upregulated in colorectal cancer tissues, and high expression of NONO was associated with poor response of radio-therapy. Moreover, compared with adjacent normal tissues, the interaction between NONO and RPLP0 was stranger in CRC tissues. NONO enhanced radio-resistance and DNA damage repair by recruiting RPLP0 to DNA damage site. High expression of NONO was a biomarker for poor treatment response of radio-therapy.