Translation controlled

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

Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control

The eukaryotic translation initiation factor 2B (eIF2B) provides a fundamental controlled point in the pathway of protein synthesis. eIF2B is the heteropentameric guanine nucleotide exchange factor that converts eIF2, from an inactive guanosine diphosphate–bound complex to eIF2-guanosine triphosphate. This reaction is controlled in response to a variety of cellular stresses to allow the rapid reprogramming of cellular gene expression. Here we demonstrate that in contrast to other translation initiation factors, eIF2B and eIF2 colocalize to a specific cytoplasmic locus. The dynamic nature of this locus is revealed through fluorescence recovery after photobleaching analysis. Indeed eIF2 shuttles into these foci whereas eIF2B remains largely resident. Three different strategies to decrease the guanine nucleotide exchange function of eIF2B all inhibit eIF2 shuttling into the foci. These results implicate a defined cytoplasmic center of eIF2B in the exchange of guanine nucleotides on the eIF2 translation initiation factor. A focused core of eIF2B guanine nucleotide exchange might allow either greater activity or control of this elementary conserved step in the translation pathway

Cellular eIF2B subunit localisation: implications for the integrated stress response and its control by small molecule drugs

eIF2 is a G protein critical for translation. It is tightly regulated in the integrated stress response (ISR) via phosphorylation of eIF2α and the subsequent control of eIF2B, a multisubunit guanine nucleotide exchange factor (GEF). Through studying the localisation of eIF2B subunits we identified cytoplasmic eIF2B bodies in mammalian cells. We highlight a relationship between body size and the eIF2B subunits localising to them; larger bodies contain all subunits and smaller bodies contain predominantly catalytic subunits. eIF2 localises to eIF2B bodies and shuttles within these bodies in a manner which correlates with eIF2B activity. Upon stress eIF2α-P localises predominately to larger bodies and results in a decreased shuttling of eIF2. Interestingly drugs which inhibit the ISR can rescue eIF2 shuttling in a manner correlating to levels of eIF2α-P. In contrast, smaller bodies show increased eIF2 shuttling in response to stress, which is accompanied by the localisation of eIF2Bδ to these bodies, suggesting the formation of a novel trimeric complex of eIF2B. This response is mimicked by ISR inhibiting drugs, providing insight into their potential mechanism of action. This study provides evidence that the composition and function of mammalian eIF2B bodies is regulated by the ISR and drugs which control it

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1:implications for the translational regulation of an aquaporin

Background: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5′ untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10–80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. Results: Polysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5′UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5′uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. Conclusions: Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production

Mutational analysis of the alpha subunit of eIF2B provides insights into the role of eIF2B bodies in translational control and VWM disease.

Eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within protein synthesis and regulates translation initiation in response to cellular stress. Mutations within eIF2B result in the fatal disease, leukoencephalopathy with vanishing white matter (VWM). Previous biochemical studies on VWM mutations have illustrated that changes in the activity of eIF2B poorly correlates with disease severity. This suggests that there may be additional characteristics of eIF2B contributing to VWM pathogenesis. Here, we investigated whether the localisation of eIF2B to eIF2B bodies was integral for function and whether this localisation could provide insight into the pathogenesis of VWM. We demonstrate that the regulatory subunit, eIF2Bα, is required for the assembly of eIF2B bodies in yeast and that loss of eIF2B bodies correlates with an inability of cells to regulate eIF2B activity.  Mutational analysis of eIF2Bα showed that missense mutations which disrupt the regulation of eIF2B similarly disrupt the assembly of eIF2B bodies. In contrast, when eIF2Bα mutations which impact the catalytic activity of eIF2B were analysed, eIF2B bodies were absent and instead eIF2B localised to small foci, termed microfoci. FRAP analysis highlighted that within these microfoci, eIF2 shuttles more slowly indicating that formation of eIF2B bodies correlates with full eIF2B activity. When eIF2Bα VWM mutations were analysed a diverse impact on localisation was observed, which did not seem to correlate with eIF2B activity.  These findings provide key insights into how the eIF2B body assembles and suggest that the body is a fundamental part of the translational regulation via eIF2α phosphorylation

Exceptionally stable pre-industrial sea level inferred from the western Mediterranean Sea

An accurate record of pre-industrial (pre-1900 CE) sea level is necessary to place modern global mean sea-level rise in context with respect to natural variability. We present new results from precisely dated phreatic overgrowths on speleothems (POS) that preserve a detailed history of Late Holocene sea level. These data indicate that the largest sea-level jump occurred between 0.12 and 0.31 m (95% confidence) from 3.26 to 2.84 ka BP (2σ). Our results show that relative sea level stayed within 0.08 m (95% confidence) of pre-industrial levels from 2.84 ka BP to 1900 CE. This sea-level history is consistent with models of glacial isostatic adjustment that adopt a relatively weak upper mantle viscosity of ~1020 Pa s. Models indicate virtual certainty (> 0.999 probability) that rates of sea-level rise over the past 4 ka (including the 400-year jump) have not approached the global average since 1900 CE; therefore, recent sea-level rise cannot be explained by natural variability

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