Translation controlled

A report of the meeting 'Translational Control', Cold Spring Harbor, USA, 3-7 September 2008

Change in Nutritional Status Modulates the Abundance of Critical Pre-initiation Intermediate Complexes During Translation Initiation \u3cem\u3ein Vivo\u3c/em\u3e

In eukaryotic translation initiation, eIF2∙GTP–Met-tRNAiMet ternary complex (TC) interacts with eIF3–eIF1–eIF5 complex to form the multifactor complex (MFC), while eIF2∙GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that ∼ 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, ∼ 40% of the total eIF2 is associated with eIF5 but lacks tRNAiMet. When the gcn2Δ mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNAiMet

Mechanisms of translational regulation by a human eIF5-mimic protein

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNAiMet ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses

BACKGROUND: Translation factors eIF4E and eIF4G form eIF4F, which interacts with the messenger RNA (mRNA) 5' cap to promote ribosome recruitment and translation initiation. Variations in the association of eIF4F with individual mRNAs likely contribute to differences in translation initiation frequencies between mRNAs. As translation initiation is globally reprogrammed by environmental stresses, we were interested in determining whether eIF4F interactions with individual mRNAs are reprogrammed and how this may contribute to global environmental stress responses. RESULTS: Using a tagged-factor protein capture and RNA-sequencing (RNA-seq) approach, we have assessed how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change globally in response to three defined stresses that each cause a rapid attenuation of protein synthesis: oxidative stress induced by hydrogen peroxide and nutrient stresses caused by amino acid or glucose withdrawal. We find that acute stress leads to dynamic and unexpected changes in eIF4F-mRNA interactions that are shared among each factor and across the stresses imposed. eIF4F-mRNA interactions stabilised by stress are predominantly associated with translational repression, while more actively initiating mRNAs become relatively depleted for eIF4F. Simultaneously, other mRNAs are insulated from these stress-induced changes in eIF4F association. CONCLUSION: Dynamic eIF4F-mRNA interaction changes are part of a coordinated early translational control response shared across environmental stresses. Our data are compatible with a model where multiple mRNA closed-loop complexes form with differing stability. Hence, unexpectedly, in the absence of other stabilising factors, rapid translation initiation on mRNAs correlates with less stable eIF4F interactions

The Yeast La Related Protein Slf1p Is a Key Activator of Translation during the Oxidative Stress Response

The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p ‘closed loop’ complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control

Less translational control, more memory

A small molecule can enhance the memories of rats and mice by blocking the integrated stress response in these animals

Enhanced translation initiation factor 4G levels correlate with production levels of monoclonal antibodies in recombinant CHO cell lines

Using cells to manufacture protein-based therapeutics or biopharmaceuticals is a rapidly expanding industrial activity. Chinese hamster ovary (CHO) cells are the most frequently used mammalian host-expression system for the manufacture of biopharmaceuticals. Over the past ∼30 years academic and industrial researchers have studied cell expression characteristics with aims to improve product yield, quality, scalability and reproducibility. Although many steps in the gene expression and secretion pathways have been optimized, little attention has been paid to optimizing protein synthesis factors and regulators during this process. A new study in Biochemical Journal by Mead et al., provides a first systematic study of several protein synthesis factors and finds that the expression level of eIF4G1 correlates with the level of recombinant protein expressed in cultures. Optimizing levels and activities of protein synthesis factors may help to enhance recombinant protein expression of biopharmaceuticals.</jats:p

Identification of domains and residues within the ε subunit of eukaryotic translation initiation factor 2B (eIF2Bε) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bɛ). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bɛ in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2Bɛ (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bɛ (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bɛ without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bɛ disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bɛ alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bɛ alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bɛ and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bɛ is an activation domain that responds to eIF2B complex formation