Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5' UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of labeled and unlabeled mRNA recruitment factors on both the rate and factor dependence of mRNA recruitment to the translation preinitiation complex in response to RNA sequence-and structure-changes. In addition to providing streamlined and detailed protocols, we provide a new construct for purification of higher yields of fluorescently labeled and unlabeled full-length eIF4G.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands-on laboratory exercise incorporating the use of CRISPR-Cas9 and homology directed repair (HDR) to edit two well-characterized genes in the budding yeast, Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow-up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type.

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.

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA binding. Recent evidence suggests mRNAs have variable dependence on eIF4B, suggesting this factor could promote changes in mRNA selection to adapt to stressors.However, the importance of eIF4B and its constituent domains for mRNA selection under diverse cellular and environmental conditions remain undefined. Here we compared the effects of disrupting eIF4B RNA-and ribosome-binding under ~1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but ribosome binding by the N-terminal Domain (NTD) promoted growth in response to various stressors. In particular, the NTD conferred a strong growth advantage in the presence of urea. Ribosome profiling revealed that the NTD promoted translation of mRNAs with long and highly structured 5-prime untranslated regions, both with and without urea exposure. Because these changes required 40S binding, our results suggest eIF4B regulates mRNA loading and scanning as a part of the PIC, rather than by activating mRNPs prior to ribosome binding. Furthermore, our data indicate the yeast response to urea includes a translational component, driven by translation of mRNAs encoding proteins associated with the cellular periphery, suggesting general eIFs can promote diverse cellular responses.

The eukaryotic translation initiation factor 4A (eIF4A) supports mRNA recruitment to the ribosomal preinitiation complex through activity of its conserved DEAD‐box RNA helicase motif. Interestingly, in plants eIF4A is differentially phosphorylated near the DEAD motif during various phases of the cell cycle by CDKA, lowering translation activity during mitosis ( Bush et al., 2016). The same Threonine residue of yeast eIF4A (T146) is present in a consensus CDK1/CDKA/Cdc28 motif, and is highly phosphorylated in large‐scale studies ( Soulard et al., 2010). To further dissect the molecular function of eIF4A, we are analyzing specific in vitro and in vivo changes that take place due to phosphorylation of eIF4A under normal conditions and in response to stress. We have mutated eIF4A residue T146 to phosphodeficient (Ala) and phosphomimetic (Glu, Asp) forms, and analyzed the effects of these mutations on growth and translation. Our data indicate that the phosphorylation status of eIF4A sensitizes yeast cells to membrane stressors in a manner affected by eIF4A•eIF4B coupling, and that constitutive phosphorylation of this residue is lethal due to arrest prior to cell division. Mechanistically, phosphorylation disrupts RNA binding by eIF4A. Together our results suggest that dynamic phosphorylation of eIF4A modulates translation changes that promote cell division and resistance to external stressors. Support or Funding Information This work was supported by NIH grant R00GM119173 and startup funds from SUNY at Buffalo‐College of Arts and Sciences A.SoulardA.CremonesiS.MoesF.SchützP.JenöM. N.HallMolecular biology of the cell211934753486C. P.AlbuquerqueH.ZhouMolecular & Cellular Proteomics7713891396A. O.HelbigS.RosatiP. W.PijnappelB.van BreukelenM. H.TimmersS.MohammedA. J.HeckBMC genomics111685M. S.BushO.PierratC.NibauV.MikitovaT.ZhengF. M.CorkeJ. H.DoonanPlant physiology1721128140F.ZhouS. E.WalkerS. F.MitchellJ. R.LorschA. G.HinnebuschJBC289317041722