The FGF2 gene encodes the basic fibroblast growth factor which controls cellular growth, proliferation, and cell signaling. Under normal conditions, FGF2 mRNA is translated via cap-dependent translation. However, under certain stress conditions, an internal ribosomal entry site (IRES) reportedly bypasses typical regulation switching to cap-independent translation. Cancer cells overexpress FGF2 protein, ultimately leading to tumor growth. Mechanistically, the reported switch from cap-dependent to cap-independent translation remains poorly described. In order to understand the regulation of the FGF2 IRES under cellular stress conditions, we are investigating its secondary structure using the SHAPE-MaP (selective 2ʹ-hydroxyl acylation analyzed by primer extension with mutation profiling) chemical probing strategy under different chemically-induced stress factors. Here we describe our experimental strategy and present a secondary structure model of the FGF2 mRNA 5ʹ UTR built using gently extracted total RNA from the A549 human lung carcinoma cell line, using phenol:chloroform:isoamyl alcohol. We are observing a complex arrangement of nucleotides throughout the targeted region (nucleotides 2-426). We are comparing the cell-free SHAPE reactivity profile with reactivity data from live cells using reagents 5NIA. Prior studies show that FGF2 uses four upstream CUG start codons which are activated in transformed and stressed cells. We present our initial reports on stress-induced cells, where we compare SHAPE profiles to identify the effect of stressors such as hypoxia, nutrient starvation, and heat shock. We aim to elucidate structural mechanisms corresponding to the effects of stress on FGF2 translation control. Lastly, we describe a workflow for future experiments involving DMS-MaP, a strategy for direct nucleotide base-pairing analysis, and RNP-MaP, an RNA-protein interactomics strategy to study the influence of IRES-transacting factors under stress conditions.


GCSU Dept. of Biological and Environmental Sciences, NSF

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