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

Alessandra Bisio,Giovanna Bianchi Scarra,Robert C Spitale,Elisa Latorre,Virginia Andreotti,Alberto Inga,Alessandro Provenzani,Mark Harland,Paola Ghiorzo,Brigitte Bressac-De Paillerets

doi:10.18632/oncotarget.5387

Abstract

CDKN2A/p16INK4a 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 p16INK4a 5'UTR mRNA acts as a cellular Internal Ribosome Entry Site (IRES). The potential for p16INK4a 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 p16INK4a 5'UTR could stimulate cap-independent translation. Notably, hypoxic stress and the treatment with mTOR inhibitors enhanced the translation-stimulating property of p16INK4a 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 p16INK4a 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 p16INK4a 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 p16INK4a 5'UTR contains a cellular IRES that can enhance mRNA translation efficiency, in part through YBX1.

Highlights

Cap-dependent ribosome recruitment to mRNAs is the prevalent translation initiation process [1, 2] but cells are able to shift to a cap-independent mechanism in response to specific environmental conditions and cellular stresses [3,4,5]
We demonstrate that a germline sequence variant found in the p16INK4a 5′UTR (c.-42T>A) of a multiple primary melanoma patient results in local flexibility changes in RNA structure, impairing the binding of Y-box binding protein 1 (YBX1) and its stimulatory effect on Internal Ribosomal Entry Sites (IRES)-dependent translation efficiency
A panel of bicistronic reporters where the full-length p16INK4a 5′UTR or two different deletion fragments cloned as intervening sequences between Renilla and Firefly luciferase genes were used for transient transfection assays in MCF7 cells (Figure 1A)

Summary

INTRODUCTION

Cap-dependent ribosome recruitment to mRNAs is the prevalent translation initiation process [1, 2] but cells are able to shift to a cap-independent mechanism in response to specific environmental conditions and cellular stresses [3,4,5]. We demonstrate that p16INK4a 5′UTR acts as a cellular IRES and we discovered YBX1 as a positive regulator of p16INK4a cap-independent translation under hypoxic stress both in cancer-derived cell lines and p16INK4a wild type lymphoblastoid cells obtained from a melanoma patient. We demonstrate that a germline sequence variant found in the p16INK4a 5′UTR (c.-42T>A) of a multiple primary melanoma patient results in local flexibility changes in RNA structure, impairing the binding of YBX1 and its stimulatory effect on IRES-dependent translation efficiency. This sequence variant appears to alter p16 protein expression. Impaired p16 translation under hypoxia could provide a mechanistic clue to explain melanomagenesis associated with this germline variant

RESULTS

DISCUSSION

MATERIALS AND METHODS