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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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Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs
mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5′ untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3–6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5′ UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs.
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- Award ID(s):
- 2330451
- PAR ID:
- 10574066
- Publisher / Repository:
- Cell Press
- Date Published:
- Journal Name:
- Molecular Cell
- Volume:
- 85
- Issue:
- 2
- ISSN:
- 1097-2765
- Page Range / eLocation ID:
- 445 to 459.e5
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.molcel.2024.11.030
