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ROLE OF REGULATORY MRNA STRUCTURES IN THE EXPRESSION OF CANCER-RELATED GENES**

Abstract

Messenger RNA (mRNA) translation, the synthesis of proteins based on genetic code, is an essential process in gene expression. Translation is often inhibited when cells are under stress, but there are mechanisms that allow certain genes to bypass stress-induced inhibition. Prior studies have identified a list of genes that disregard stress signals to continue mRNA translation using regulatory RNA structures to hijack the translational machinery. We hypothesize that stress-signals must alter the RNA structure to trigger structure-mediated mRNA translation. Our target genes include oncogenes HIF1A, FGF2, and tumor-suppressor gene TP53. We aim to amplify regulatory mRNA regions previously reported for each gene from A549 lung carcinoma cell lines. Next, we will build and compare structure models for regulatory regions under three conditions: (a) cell-free, (b) in-cell unstressed, and (c) in-cell stressed. For structure modeling we use SHAPE-MaP, a chemical-probing strategy analyzed using next-generation sequencing (NGS). We have successfully extracted, probed, and amplified the target regions using gene-specific primers, reverse transcription (RT), and polymerase chain reaction (PCR). We analyzed PCR products using gel electrophoresis. Here we describe our experimental workflow along with data from NGS library preparation. Applying NGS using the Illumina MiSeq platform on target mRNAs, we will build RNA secondary structures with Shapemapper2 and SuperFold software packages. Our long-term goal is to describe structure-based mechanisms by which translation of cancer-causing mRNAs can be blocked, and how translation in cancer-fighting mRNAs can be reengaged.

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