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DEFINING THE STRUCTURE AND REGULATORY POTENTIAL OF THE CIRBP 5′UTR TO UNDERSTAND ITS STRESS RESPONSE ROLE**

Abstract

Cold-Inducible RNA Binding Protein (CIRBP), a translation regulator, acts as a tumor suppressor and oncogene in various cancers. Under stress, some mRNAs bypass key steps of cap-dependent translation via Internal Ribosome Entry Sites (IRES). To explore how CIRBP maintains expression under stress, we looked at its mRNA 5′UTR. Using SHAPE-MaP (Selective 2′Hydroxyl Acetylation Analyzed by Primer Extension and Mutational Profiling), we analyzed its 5′UTR secondary structure in A549 cells to confirm it is well-structured. To observe it in a native context, we performed in-cell SHAPE-MaP. SHAPE reactivity analysis shows statistically significant protected regions, indicating RNA−protein interactions that may govern CIRBP-regulated translation. We extended this analysis to cold stress to identify stress-specific contact sites. To test for IRES activity, we used a dual luciferase reporter (DLR) assay by inserting the 5′UTR between luciferase reporters in a plasmid and transfecting it into live A549 cells . Luminescence ratios quantify its ability to initiate non-canonical translation. Here, we present our cloning strategy, validation using Nanopore whole plasmid sequencing, and initial results from the DLR assay of unstressed cells.

Acknowledgements

National Science Foundation, GCSU MURACE

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