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

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.

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

“…Polyclonal rabbit antibodies against untagged yeast eIF4A and eIF4G were described previously (22). Polyclonal anti-eIF4A antibody was further purified from 40 ml of serum by running over a 5 ml Protein A column (Acrobiosystems Inc., MA-0422-C5) for immunoprecipitation experiments.…”

“…Polysome analysis was performed as described previously (22). In brief, yeast strains were cultured in SC-Leu media at 30°C to an OD 600 of 0.5.…”

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

“…However, this requirement is not based on the eIF4A-helicase activity-enhancing role of eIF4B. Recent reports have also indicated the presence of a class of mRNAs that are hyper-dependent on eIF4B while exhibiting less dependence on eIF4G and/or eIF4A (Liu et al ., 2021; Sen et al ., 2016). The ability of eIF4B to directly interact and bind the 40S ribosomal subunit seems to be crucial for the translation of these mRNAs.…”

“…In yeast, the NTD and 7-RR have been reported to be responsible for the interaction of eIF4B with 40S, independent of the RRM domain (Walker et al ., 2013). It has been suggested that the binding of eIF4B to the 40S ribosomal subunit may promote conformational changes in the mRNA binding channel of the ribosome, making it more accessible (Liu et al ., 2021). However, there is no structural understanding of the conformational changes in the 40S upon eIF4B binding or how eIF4B helps in the recruitment of mRNAs.…”