Deletion of the N-Terminal Domain of Yeast Eukaryotic Initiation Factor 4B Reprograms Translation and Reduces Growth in Urea.

Xiaozhuo Liu,Houtan Moshiri,Sarah E Walker,Ansuman Sahoo,Qian He

doi:10.3389/fmolb.2021.787781

Xiaozhuo Liu, Houtan Moshiri + Show 3 more

Open Access

https://doi.org/10.3389/fmolb.2021.787781

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Abstract

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA recruitment. Recent evidence indicates yeast mRNAs have variable dependence on eIF4B under optimal growth conditions. Given the ability of eIF4B to promote translation as a function of nutrient conditions in mammalian cells, we wondered if eIF4B activities in translation could alter phenotypes in yeast through differential mRNA selection for translation. Here we compared the effects of disrupting yeast eIF4B RNA- and 40S-binding motifs under ∼1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but the 40S-binding N-terminal Domain (NTD) promoted growth in response to stressors requiring robust cellular integrity. In particular, the NTD conferred a strong growth advantage in the presence of urea, which may be important for pathogenesis of related fungal species. Ribosome profiling indicated that similar to complete eIF4B deletion, deletion of the NTD dramatically reduced translation, particularly of those mRNAs with long and highly structured 5-prime untranslated regions. This behavior was observed both with and without urea exposure, but the specific mRNA pool associated with ribosomes in response to urea differed. Deletion of the NTD led to relative increases in ribosome association of shorter transcripts with higher dependence on eIF4G, as was noted previously for eIF4B deletion. Gene ontology analysis indicated that proteins encoded by eIF4B NTD-dependent transcripts were associated with the cellular membrane system and the cell wall, while NTD-independent transcripts encoded proteins associated with cytoplasmic proteins and protein synthesis. This analysis highlighted the difference in structure content of mRNAs encoding membrane versus cytoplasmic housekeeping proteins and the variable reliance of specific gene ontology classes on various initiation factors promoting otherwise similar functions. Together our analyses suggest that deletion of the eIF4B NTD prevents cellular stress responses by affecting the capacity to translate a diverse mRNA pool.

Highlights

Translation initiation begins with the formation of a translation preinitiation complex (PIC) comprised of an initiator MettRNAeIF2GTP ternary complex bound to the 40S ribosomal subunit along with eIFs 1, 1A, 5 and the multisubunit eIF3
Previous work demonstrated that the N-terminal domain (NTD) of eIF4B promotes both affinity for the 40S subunit in vitro and recruitment of mRNAs to the preinitiation complex in vitro and in vivo, while the RNA-Recognition Motif (RRM) of eIF4B is dispensable in auxotrophic yeast strains (Walker et al, 2013; Zhou et al, 2014)
We compared the parallel analysis of RNA structure (PARS) scores for all yeast mRNAs versus the PARS scores for all yeast mRNAs associated with gene ontology (GO) terms for mRNAs that required eIF4B for translation in response to urea (Figures 5A–C.) This group includes: intrinsic component of the membrane; as well as transferase activity, endomembrane system, glycosylation, and mannosylation Interestingly, we found that the 5′UTRs of mRNAs associated with each of these GO terms had higher average Total PARS scores than the average yeast mRNA (Figure 5A, 5′UTR)

Summary

Introduction

Translation initiation begins with the formation of a translation preinitiation complex (PIC) comprised of an initiator MettRNAeIF2GTP ternary complex bound to the 40S ribosomal subunit along with eIFs 1, 1A, 5 and the multisubunit eIF3. EIF4A is a DEAD-box RNA helicase with activity modulated by changes in conformation upon binding to RNA, eIF4G, eIF4B, and in mammalian cells, eIF4H (Mitchell et al, 2011) This complex is thought to serve multiple purposes: 1) interactions of the 5′cap bound to eIF4E with other components of the PIC bound to eIF4G direct PIC loading to the 5′end of mRNAs, and 2) helicase activity of eIF4A melts mRNA secondary structure near the cap and throughout the 5-prime untranslated region (5′UTR) to allow effective loading at the cap and scanning through 5′UTRs. The associated protein eIF4B promotes the activity of the eIF4F complex (Rozovsky et al, 2008; Özeş et al, 2011). These eIF4A-independent functions could stem from the ability of eIF4B to bind to the 40S subunit and promote conformational changes in the mRNA binding channel (Walker et al, 2013; Sen et al, 2016)

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