Stress-induced changes in endogenous TP53 mRNA 5' regulatory region.

Abstract

Tumor suppressor protein p53 is regulated in a number of ways, including during initiation of TP53 mRNA translation. The 5' end of TP53 mRNA contains regulatory structures that enable noncanonical initiation using mechanisms that remain poorly described. Here we analyze per-nucleotide reactivity changes in the 5' end secondary structure of TP53 mRNA under in-cell conditions using A549 human lung carcinoma cells. We first construct a cell-free secondary structure model using SHAPE reagent 5-nitroisatoic anhydride on gently extracted and deproteinated RNA. We observe previously described regulatory features of the TP53 mRNA 5' end including two motifs which we refer to as long stem-loop (LSL) and short stem-loop (SSL), respectively. We observe a domain-forming helix that groups LSL and SSL, forming a three-helix junction. Applying in-cell selective 2' hydroxyl acylation analyzed by primer extension and mutational profiling, we assess reactivity profiles with unstressed cells and with chemically induced stress conditions expected to stimulate TP53 cap-independent translation. We analyze the effects of etoposide-induced DNA damage, CoCl₂-induced hypoxia, and 5' cap inhibition with 4EGI-1 treatment. Identifying stress-associated changes in the TP53 5' end may help elucidate the role of regulatory RNA structure in cap-independent translation. Using ΔSHAPE, we identify in-cell protection sites that correspond with previously described RNA-protein binding sites on the apical loops of LSL and SSL. Furthermore, we identify several other potential interaction sites, some associated with specific types of stress. Some noteworthy changes include ΔSHAPE sites proximal to the start codons, at the three-helix junction and on the domain-forming helix. We summarize potential interactions on the cell-free secondary structure model.