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

Abstract

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.