MRNA structural elements immediately upstream of the start codon dictate dependence upon eIF4A helicase activity

Joseph A Waldron,Ania Wilczynska,John Le Quesne,Martin Bushell,Laura E Ritchey,David C Tack,Sarah M Assmann,Sarah L Gillen,Philip C Bevilacqua,Ernest Turro

doi:10.1186/s13059-019-1901-2

Abstract

BackgroundThe RNA helicase eIF4A1 is a key component of the translation initiation machinery and is required for the translation of many pro-oncogenic mRNAs. There is increasing interest in targeting eIF4A1 therapeutically in cancer, thus understanding how this protein leads to the selective re-programming of the translational landscape is critical. While it is known that eIF4A1-dependent mRNAs frequently have long GC-rich 5′UTRs, the details of how 5′UTR structure is resculptured by eIF4A1 to enhance the translation of specific mRNAs are unknown.ResultsUsing Structure-seq2 and polysome profiling, we assess global mRNA structure and translational efficiency in MCF7 cells, with and without eIF4A inhibition with hippuristanol. We find that eIF4A inhibition does not lead to global increases in 5′UTR structure, but rather it leads to 5′UTR remodeling, with localized gains and losses of structure. The degree of these localized structural changes is associated with 5′UTR length, meaning that eIF4A-dependent mRNAs have greater localized gains of structure due to their increased 5′UTR length. However, it is not solely increased localized structure that causes eIF4A-dependency but the position of the structured regions, as these structured elements are located predominantly at the 3′ end of the 5′UTR.ConclusionsBy measuring changes in RNA structure following eIF4A inhibition, we show that eIF4A remodels local 5′UTR structures. The location of these structural elements ultimately determines the dependency on eIF4A, with increased structure just upstream of the CDS being the major limiting factor in translation, which is overcome by eIF4A activity.

Highlights

The RNA helicase eukaryotic initiation factor 4A1 (eIF4A1) is a key component of the translation initiation machinery and is required for the translation of many pro-oncogenic mRNAs
Measuring eIF4A mediated changes in RNA structure To determine the effect of eIF4A activity on RNA secondary structure in vivo, we measured the reactivity of cellular RNA to dimethyl sulphate (DMS) following eIF4A inhibition with hippuristanol in MCF7 cells (Fig. 1a)
In order to primarily inhibit the translation of eIF4Adependent mRNAs, rather than to completely ablate global translation, MCF7 cells were treated with hipp for 1 h at the IC50, as determined by 35S protein labeling (Additional file 1: Figure S1A)

Summary

Introduction

The RNA helicase eIF4A1 is a key component of the translation initiation machinery and is required for the translation of many pro-oncogenic mRNAs. There is increasing interest in targeting eIF4A1 therapeutically in cancer, understanding how this protein leads to the selective re-programming of the translational landscape is critical. The helicase activity of eIF4A is relatively weak compared with other RNA helicases [15], and it may have additional ATPase-dependent but helicase-independent roles, such as remodeling of protein/RNA complexes. Both human eIF4A1 and yeast eIF4A have been shown to enhance ribosome recruitment onto RNAs lacking secondary structure, implicating a helicase-independent role for eIF4A during translation initiation [16, 17]. Whether excess eIF4A acts as part of the translational machinery or as “free” eIF4A1 is not known, and as such, the consequence of increased levels of eIF4A1 protein, as seen in tumor cells [4], is not clear

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