The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A

Querido, Jailson Brito; Sokabe, Masaaki; Díaz-López, Irene; Gordiyenko, Yuliya; Fraser, Christopher S.; Ramakrishnan, V.

doi:10.1101/2022.12.07.519490

Cited by 8 publications

(15 citation statements)

References 89 publications

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“…Recombinant full-length eIF4G is notoriously difficult to purify and possesses potent non-specific RNA binding activity 30,35 . Consequently, many biochemical studies have been performed with truncated eIF4G constructs 14,15,19,23,24,26,[36][37][38][39] together with short, minimal-length mRNA mimics to minimize nonspecific binding 15,24,30,38,39 . Biochemical studies have also been hampered by the lack of a complete structure of eIF4F 13,14,27,38 .…”

Section: Introductionmentioning

confidence: 99%

The mechanism of mRNA activation

Gentry,

Ide,

Comunale

et al. 2023

Preprint

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SummaryDuring translation initiation, messenger RNA molecules must be identified and activated for loading into a ribosome. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5’ end of the messenger RNA and subsequently activate the message. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and messenger RNA activation remain mysterious. Here, we generate a unique, single-molecule fluorescence imaging system to interrogate the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native messenger RNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the messenger RNA and identify allosteric communication between the eIF4F subunits which ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover novel functions for each subunit of eIF4F and we conclude by presenting a model for messenger RNA activation which precisely defines the composition of the activated message. This model provides a general framework for understanding how messenger RNA molecules may be discriminated from one another, and how other RNA-binding proteins may control the efficiency of translation initiation.

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Section: Introductionmentioning

confidence: 99%

The mechanism of mRNA activation

Gentry,

Ide,

Comunale

et al. 2023

Preprint

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“…While we were finalizing this manuscript a structure of human 48S PIC was reported where a low-resolution density in the vicinity of the mRNA channel entry on the solvent-exposed side is tentatively assigned to eIF4B (Brito Querido et al, 2022). Overall, the structural analysis presented in this study shows how yeast eIF4B helps in mRNA recruitment as well as scanning during translation initiation.…”

Section: Discussionmentioning

confidence: 74%

“…Based on our observations and comparison of 40S-eIF4B with reported structures of partial yeast 43S and 48S PICs, were have proposed a model explaining the events that may happen during mRNA recruitment to the small ribosomal subunit in yeast (Figure 5). While we were finalizing this manuscript a structure of human 48S PIC was reported where a low-resolution density in the vicinity of the mRNA channel entry on the solvent-exposed side is tentatively assigned to eIF4B (Brito Querido et al ., 2022). Overall, the structural analysis presented in this study shows how yeast eIF4B helps in mRNA recruitment as well as scanning during translation initiation.…”

Section: Resultsmentioning

confidence: 99%

Yeast eukaryotic initiation factor 4B remodels the mRNA entry site on the small ribosomal subunit

Datey

Khaja

Rahil

et al. 2023

Preprint

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Eukaryotic initiation factor 4B (eIF4B) belongs to the eIF4 group of factors that help in mRNA recruitment to the ribosomal preinitiation complex (PIC) in all eukaryotic organisms. eIF4B stimulates the helicase activity of eIF4A and helps in the formation of the 48S PIC by facilitating mRNA recruitment. However, there is no clear understanding of the location of eIF4B on the 40S and how eIF4B helps in the recruitment of mRNAs. In this work using cryo-electron microscopy, we show that yeast eIF4B binds to the 40S ribosomal subunit at the mRNA entry channel making contacts with ribosomal proteins uS10, uS3, and eS10 and ribosomal rRNA helix h16. The yeast eIF4B position on the 40S overlaps with the RRM domain of eIF3g indicating that the binding of eIF4B may trigger the relocation of the eIF3 b-g-i module to the subunit interface. The 40S head is in partially open conformation that may facilitate the release of eIF3j and hence aid mRNA recruitment and scanning. The structural analysis of yeast eIF4B-bound ribosomal complex provides insight into possible events during mRNA recruitment.

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“…We found that eIF4A and ASCC3 regulate similar transcripts and phenotypic exacerbation was observed in response to the loss of ASCC3 in combination with eIF4A depletion. Previous cryo‐EM analysis has shown that eIF4A functions at a position near the mRNA exit channel of the 40S ribosome subunit (Querido et al , 2020), but a recent study has identified a second eIF4A that binds to the mRNA entry channel, in addition to the one that is part of eIF4F, which likely functions to unwind the secondary structure of mRNAs that enter the mRNA entry channel during scanning (preprint: Querido et al , 2022). The low helicase activity of eIF4A compared with many other helicases suggested that additional helicases may be required to scan the 5′‐UTR of mRNAs with highly stable secondary structures, and ASCC3 may be one of them for a subset of transcripts.…”

Section: Discussionmentioning

confidence: 99%

“…C Metagene plots for full-length footprints obtained by TCP-seq analysis of the SSU fraction prepared from control, eIF4A1/A2 dKD, and ASCC3 KD cells. binds to the mRNA entry channel, in addition to the one that is part of eIF4F, which likely functions to unwind the secondary structure of mRNAs that enter the mRNA entry channel during scanning (preprint: Querido et al, 2022). The low helicase activity of eIF4A compared with many other helicases suggested that additional helicases may be required to scan the 5 0 -UTR of mRNAs with highly stable secondary structures, and ASCC3 may be one of them for a subset of transcripts.…”

Section: Discussionmentioning

confidence: 99%

The ASC‐1 complex promotes translation initiation by scanning ribosomes

et al. 2023

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Translation initiates when the eIF4F complex binds the 5 0 mRNA cap, followed by 5 0 untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP-seq) analysis revealed that ASCC3, a helicase domaincontaining subunit of ASCC, localizes predominantly to the 5 0 untranslated region of mRNAs. Ribo-seq, TCP-seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.

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The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A

Cited by 8 publications

References 89 publications

The mechanism of mRNA activation

The mechanism of mRNA activation

Yeast eukaryotic initiation factor 4B remodels the mRNA entry site on the small ribosomal subunit

The ASC‐1 complex promotes translation initiation by scanning ribosomes

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