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5'-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs.

ABSTRACT: During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

SUBMITTER: Haizel SA

PROVIDER: S-EPMC7450097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Utilization of alternative initiation sites for protein translation directed by non-AUG codons in mammalian mRNAs is observed with increasing frequency. Alternative initiation sites are utilized for the synthesis of important regulatory proteins that control distinct biological functions. It is, therefore, of high significance to define the parameters that allow accurate bioinformatic prediction of alternative translation initiation sites (aTIS). This study has investigated 5'-UTR regions of mRNAs to define consensus sequence properties and structural features that allow identification of alternative initiation sites for protein translation. RESULTS: Bioinformatic evaluation of 5'-UTR sequences of mammalian mRNAs was conducted for classification and identification of alternative translation initiation sites for a group of mRNA sequences that have been experimentally demonstrated to utilize alternative non-AUG initiation sites for protein translation. These are represented by the codons CUG, GUG, UUG, AUA, and ACG for aTIS. The first phase of this bioinformatic analysis implements a classification tree that evaluated 5'-UTRs for unique consensus sequence features near the initiation codon, characteristics of 5'-UTR nucleotide sequences, and secondary structural features in a decision tree that categorizes mRNAs into those with potential aTIS, and those without. The second phase addresses identification of the aTIS codon and its location. Critical parameters of 5'-UTRs were assessed by an Artificial Neural Network (ANN) for identification of the aTIS codon and its location. ANNs have previously been used for the purpose of AUG start site prediction and are applicable in complex. ANN analyses demonstrated that multiple properties were required for predicting aTIS codons; these properties included unique consensus nucleotide sequences at positions -7 and -6 combined with positions -3 and +4, 5'-UTR length, ORF length, predicted secondary structures, free energy features, upstream AUGs, and G/C ratio. Importantly, combined results of the classification tree and the ANN analyses provided highly accurate bioinformatic predictions of alternative translation initiation sites. CONCLUSION: This study has defined the unique properties of 5'-UTR sequences of mRNAs for successful bioinformatic prediction of alternative initiation sites utilized in protein translation. The ability to define aTIS through the described bioinformatic analyses can be of high importance for genomic analyses to provide full predictions of translated mammalian and human gene products required for cellular functions in health and disease.

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5'-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs.

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