In mammalian systems, controlled progression through the cell cycle is essential for normal proliferation and its loss is a hallmark of malignancy. p16INK4a inhibits the cyclin-dependent kinases CDK4 and CDK6, thereby keeping the retinoblastoma protein (pRB) in a hypo-phosphorylated state, which can lead to G1/S checkpoint activation. In addition p16INK4a plays a crucial role in the process of replicative senescence. p16INK4a loss or inactivation is associated with predisposition to melanoma, pancreatic cancer and other malignancies, highlighting its recognized function as a tumor suppressor. Given its critical role in cell homeostasis, there is much interest in understanding the molecular regulators of p16INK4a expression.

Here we report that p16 belongs to the expanding group of proteins whose translation is influenced by sequence/structural features of the 5’UTR mRNA that are endowed of cellular Internal Ribosome Entry Site (IRES) activity. To study the potential for p16INK4a 5’UTR to drive cap-independent translation we developed a dual-luciferase assay using a bicistronic vector (named pRuF), where wild type or deletion mutants of the p16INK4a 5’UTRs were cloned as intervening sequence between Renilla and Firefly luciferase cDNAs. The p16INK4a 5’UTR sequence in inverted orientation was included as additional control. Results of reporters’ relative activity coupled to control analyses of actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5’UTR could stimulate Firefly luciferase translation. The IRES-like activity could not be mapped to a specific region of the 5’UTR based on results with deletion constructs, and was two-fold stronger compared to the cMYC 5’UTR, a known cellular IRES used as a control. Notably, hypoxic stress in particular, but also the treatment with mTOR inhibitors, enhanced the translation-stimulating property of the p16INK4a 5’UTR. RNA immuno-precipitation (RIP) assays performed in the p16-positive melanoma-derived cell line SK-Mel-28 suggest that the RNA-binding protein YBX-1, known to act in translation control, can participate in p16 INK4a translation. Experiments were YBX-1 was over-expressed or knocked-down by si-RNA confirmed its involvement in p16INK4a cap-independent translational regulation. Taken collectively, our results suggest that the 5’UTR region can modulate p16INK4a mRNA translation efficiency.

Citation Format: Alessandra Bisio, Elisa Latorre, Virginia Andreotti, Alessandro Provenzani, Giovanna Bianchi- Scarrà, Paola Ghiorzo, Alberto Inga. An internal ribosomal entry site in the 5’-untranslated region of p16INK4a mRNA provides a novel mechanism for the regulation of its translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3384. doi:10.1158/1538-7445.AM2014-3384