Project description:The aim of this study is to investigate how eIF2 phosphorylation affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts.
Project description:In times of cellular stress, such as during virus infections, the integrated stress response (ISR) blocks translation initiation through phosphorylation of the essential translation initiation factor eIF2. Phosphorylated eIF2 (p-eIF2) sequesters the eIF2-specific guanidine exchange factor (GEF) eIF2B, thereby preventing eIF2 recycling. Here we describe the first example of a viral ISR antagonist that inhibits the ISR at its most central step: the interplay between p-eIF2 and eIF2B. Using AP-MS, we determine that BW10 binds eIF2B. There, it selectively displaces eIF2B’s inhibitor p-eIF2 without affecting the association of its substrate eIF2. By this mechanism, BW10 renders cellular translation immune to regulation by eIF2 phosphorylation. Thus, under stress conditions BW10 creates the unprecedented situation of high levels of p-eIF2 coinciding with unimpaired translation.
Project description:Disruption of protein folding in the endoplasmic reticulum triggers the Unfolded Protein Response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PERK phosphorylation of the alpha subunit of eIF2 (eIF2~P), which represses global translation coincident with preferential translation of mRNAs, such as ATF4 and CHOP, that serve to implement the UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKa as being subject to both translation and transcriptional induction during eIF2~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKalpha mRNA involves the stress-induced relief of two inhibitory uORFs in the 5'-leader of the transcript. Depletion of IBTKalpha by shRNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKalpha is a key regulator in determining cell fate during the UPR. We used a genome-wide microarray approach to determine how individual mRNAs were differentially translated during endoplasmic reticulum stress.
Project description:mRNA translation plays a major role in homeostasis, whereas its dysregulation underpins a variety of pathological states including cancer, metabolic syndrome and neurological disorders. Ternary complex (TC) and eIF4F complex assembly are two major rate-limiting steps in translation initiation that are thought to be regulated by eIF2α phosphorylation, and the mTOR/4E-BP pathway, respectively2. However, how TC and eIF4F assembly are coordinated remains largely unknown. Using polysome-profiling, we show that on a genome-wide scale mTOR suppresses translation of mRNAs, which are translationally activated under short-term ER stress when TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation, which increases recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β appears to act as a previously unidentified mediator of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.
Project description:Disruption of protein folding in the endoplasmic reticulum triggers the Unfolded Protein Response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PERK phosphorylation of the alpha subunit of eIF2 (eIF2~P), which represses global translation coincident with preferential translation of mRNAs, such as ATF4 and CHOP, that serve to implement the UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKa as being subject to both translation and transcriptional induction during eIF2~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKalpha mRNA involves the stress-induced relief of two inhibitory uORFs in the 5'-leader of the transcript. Depletion of IBTKalpha by shRNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKalpha is a key regulator in determining cell fate during the UPR. We used a genome-wide microarray approach to determine how individual mRNAs were differentially translated during endoplasmic reticulum stress. A microarray analysis from our laboratory identified gene transcripts suggested to be under translation control in mouse embryonic fibroblast (MEF) cells following a 6 hour treatment with thapsigargin, a potent inducer of ER stress, or no stress. The mRNAs were separated by sucrose gradient analyses to yield three fractions, those transcripts associated with large polysomes (?4 ribosomes per mRNA), those associated with monosome, disomes, or trisomes, and those fractionated at the top of the gradient with free ribosomes. RNA was extracted from sucrose gradients corresponding to these fractions and hybridized on Affymetrix microarrays. In parallel, we also measured total levels for each gene transcript in the presence or absence of thapsigargin treatment to address transcription regulation coincident with translational control. Please note that the treatment plus fractionation based on association with different numbers of ribosomes did yield different populations of mRNAs, which resulted in considerable variation in normalized data across the samples.
Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. This SuperSeries is composed of the following subset Series:; GSE11496: Expression data from Gcn2 wild-type and knockout mouse liver perfused with or without methionine; GSE11684: Expression data from Perk wild-type and knockout mouse liver perfused without or with 2,5-di-tert-butylhydroquinone Experiment Overall Design: Refer to individual Series
Project description:Tumor-associated macrophages (TAMs) continuously tune their immune modulatory properties, but how gene expression programs coordinate this is largely unknown. Selective mRNA translation facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs and plays pivotal roles in regulating immune cell plasticity. Using polysome-profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies targeting key cellular functions including cell proliferation and metabolism. These changes in translational efficiencies paralleled accumulation of immunosuppressive macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, whose altered phosphorylation selectively modulates mRNA translation. Furthermore, suppression of MNK2-signaling reprogrammed immunosuppressive macrophages towards a pro-inflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2-signaling represents a key node regulating macrophage immunosuppressive functions.