The 5′-untranslated region of p16INK4a melanoma tumor suppressor acts as a cellular IRES, controlling mRNA translation under hypoxia through YBX1 binding

CDKN2A/p16(INK4a) is an essential tumor suppressor gene that controls cell cycle progression and replicative senescence. It is also the main melanoma susceptibility gene. Here we report that p16(INK4a) 5'UTR mRNA acts as a cellular Internal Ribosome Entry Site (IRES). The potential for p16(INK4a) 5'UTR to drive cap-independent translation was evaluated by dual-luciferase assays using bicistronic and monocistronic vectors. Results of reporters' relative activities coupled to control analyses for actual bicistronic mRNA transcription, indicated that the wild type p16(INK4a) 5'UTR could stimulate cap-independent translation. Notably, hypoxic stress and the treatment with mTOR inhibitors enhanced the translation-stimulating property of p16(INK4a) 5'UTR. RNA immunoprecipitation performed in melanoma-derived SK-Mel-28 and in a patient-derived lymphoblastoid cell line indicated that YBX1 can bind the wild type p16(INK4a) mRNA increasing its translation efficiency, particularly during hypoxic stress. Modulation of YBX1 expression further supported its involvement in cap-independent translation of the wild type p16(INK4a) but not a c.-42T>A variant. RNA SHAPE assays revealed local flexibility changes for the c.-42T>A variant at the predicted YBX1 binding site region. Our results indicate that p16(INK4a) 5'UTR contains a cellular IRES that can enhance mRNA translation efficiency, in part through YBX1.