Abstract B09: hnRNPA1 is a regulator of UNR IRES activity

Alternative translation initiation via internal ribosome entry sites (IRESs) allows for sustained expression of specific proteins under conditions, such as stress, where global translation is inhibited. As cells in tumors are constantly facing stress conditions, e.g. hypoxia or nutrient deprivation, it is not surprising that mRNAs encoding for oncogenes appear to be enriched in IRESs. IRES-dependent translation in eukaryotes commonly involves certain RNA-binding proteins, the so-called IRES trans-acting factors (ITAFs), that can directly interact with the IRESs to facilitate translation initiation. Upstream of N-Ras (UNR) is such an ITAF that can regulate the IRES activity of several cellular and viral IRESs. It is known that its 59 untranslated region (59 UTR) also contains an IRES that is regulated by polypyrimidine-tract binding protein (PTB), heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 and UNR itself. Prediction analyses indicated that hnRNPA1 might bind to the 59 UTR of UNR as well. hnRNPA1 was previously already shown to serve as an ITAF in the regulation of the IRES activities of numerous mRNAs including egr2 and sST2. The aims of this project were to determine the impact of hnRNPA1 on the IRES activity of UNR. To test, if hnRNPA1 interacts with the 59 UTR of UNR, as predicted, we employed RNA affinity chromatography. We found that hnRNPA1 indeed strongly bound to the UNR 59 UTR. To assess, if hnRNPA1 consequently affects UNR IRES activity, we overexpressed hnRNPA1. Overexpression of hnRNPA1 markedly increased UNR IRES activity. hnRNPA1 overexpression further induced total UNR protein expression, which indicates that elevated UNR IRES activity results in enhanced translation. With respect to the exact interaction sites, hnRNPA1 binding was predicted to occur at overlapping sites to the previously published PTB binding sites. Deletion of the hnRNPA1 binding sites not only decreased the UNR IRES activity, it further rendered it insensitive to elevated hnRNPA1 levels. Taken together, we found that the 59 UTR of UNR contains specific hnRNPA1 binding sites and that binding of hnRNPA1 at these sites enhances the UNR IRES activity. Our findings therefore add a new piece to the complex puzzle of UNR regulating ITAFs. Having established hnRNPA1 as a new regulator of UNR IRES activity, might further put hnRNPA1 forward as an interesting target to regulate UNR expression. Citation Format: Sebastian Lampe, Thilo Braus, Michael Kunze, Anica Scholz, Sofia Winslow, Bernhard Brune, Tobias Schmid. hnRNPA1 is a regulator of UNR IRES activity. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B09.