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Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5′UTR, and initiate cap-independent translation using elements called cap-independent translation enhancers or internal ribosome entry sites within their 5′UTRs. The interaction among initiation factors such as eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4GI, especially in regulating the eIF4F complex during noncanonical translation initiation of different 5′UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, CD studies and in vitro translation assays were used to elucidate the recruitment of these initiation factors to the highly structured 5′UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI’s binding affinity to the uncapped 5′UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5′UTR of FGF-9 mRNA. Recently, Izidoro et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here, we show that structured 5′UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs used by cells to mitigate cellular stress conditions.
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This content will become publicly available on April 1, 2026
Stress-induced changes in endogenous TP53 mRNA 5′ regulatory region
Tumor suppressor protein p53 is regulated in a number of ways, including during initiation of TP53 mRNA translation. The 50 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 50 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 50 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 20 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 50 cap inhibition with 4EGI-1 treatment. Identifying stress-associated changes in the TP53 50 end may help elucidate therole of regulatory RNA structure in cap-independent translation. Using DSHAPE, 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 DeltaSHAPE 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.
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- Award ID(s):
- 2310684
- PAR ID:
- 10586841
- Editor(s):
- Musier-Forsyth, Karin
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- Volume:
- 301
- Issue:
- 4
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- 108418
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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