Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner
Abstract
DEAD-box RNA helicase Ded1 is thought to resolve secondary structures in mRNA 5′-untranslated regions (5′-UTRs) that impede 48S preinitiation complex (PIC) formation at the initiation codon. We reconstituted Ded1 acceleration of 48S PIC assembly on native mRNAs in a pure system, and recapitulated increased Ded1-dependence of mRNAs that are Ded1-hyperdependent in vivo. Stem-loop (SL) structures in 5′-UTRs of native and synthetic mRNAs increased the Ded1 requirement to overcome their intrinsically low rates of 48S PIC recruitment. Ded1 acceleration of 48S assembly was greater in the presence of eIF4F, and domains mediating one or more Ded1 interactions with eIF4G or helicase eIF4A were required for efficient recruitment of all mRNAs; however, the relative importance of particular Ded1 and eIF4G domains were distinct for each mRNA. Our results account for the Ded1 hyper-dependence of mRNAs with structure-prone 5′-UTRs, and implicate an eIF4E·eIF4G·eIF4A·Ded1 complex in accelerating 48S PIC assembly on native mRNAs.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-6 and related figure supplements.
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Author details
Funding
National Institutes of Health
- Jon R Lorsch
- Alan G Hinnebusch
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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