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

// Alessandra Bisio 1 , Elisa Latorre 2 , Virginia Andreotti 3 , Brigitte Bressac-de Paillerets 4 , Mark Harland 5 , Giovanna Bianchi Scarra 3 , Paola Ghiorzo 3 , Robert C. Spitale 6 , Alessandro Provenzani 2 , Alberto Inga 1 1 Laboratory of Transcriptional Networks, Centre for Integrative Biology, CIBIO, University of Trento, Trento, Italy 2 Laboratory of Genomic Screening, Centre for Integrative Biology, CIBIO, University of Trento, Trento, Italy 3 Laboratory of Genetics of Rare Hereditary Cancers, DiMI, University of Genoa, Italy and IRCCS AOU San Martino-IST, Genoa, Italy 4 Service de Génétique, Institut Gustave Roussy, Villejuif, France 5 Section of Epidemiology and Biostatistics, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK 6 Department of Pharmaceutical Sciences, University of California, Irvine, CA, USA Corresponding to: Alberto Inga e-mail: inga@science.unitn.it Alessandra Bisio e-mail: bisio@science.unitn.it Keywords: p16 INK4a , YBX1, melanoma, IRES, hypoxia Received: July 14, 2015      Accepted: October 05, 2015      Published: October 17, 2015 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.