Principles of ER Co-Translational Translocation Revealed by Proximity-Specific Ribosome Profiling
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ABSTRACT:
ORGANISM(S): Homo sapiens Saccharomyces cerevisiae
PROVIDER: GSE61012 | GEO | 2014/11/10
SECONDARY ACCESSION(S): PRJNA260073
REPOSITORIES: GEO
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Project description:Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating Translocation-Associated Quality Control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. We conduct a genome-wide CRISPR/Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon, and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.
2023-01-20 | GSE222104 | GEO
Project description:In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a role for the ER in global protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression. HEK293 cells were fractionated between the cytosol and endoplasmic reticulum. Within each fraction, ribosome footprints were generated and sequenced. In parallel, total mRNA was sequenced.
2011-12-20 | E-GEOD-31539 | biostudies-arrayexpress
Project description:The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome . It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. While individual components of this machine have been studied at high resolution in isolation, insights into their interplay in the native membrane remain limited. Here, we use electron cryo-tomography (cryo-ET), extensive classification and molecular modeling to capture molecular resolution snapshots of mRNA translation and protein maturation at the ER membrane. Comprehensively recapitulating the translational elongation cycle, we identify a highly abundant classical pre-translocation (PRE) intermediate with eEF1a in an extended conformation following the decoding state, which indicates that eEF1a remains ribosome-associated after GTP-hydrolysis during proofreading. The complete atomic structure of the most abundant ER translocon variant comprising the protein-conducting channel Sec61, the translocon-associated protein complex (TRAP) and the oligosaccharyltransferase complex A (OSTA) reveals the molecular framework for signal peptide (SP) membrane insertion and protein N-glycosylation. Associated with OSTA we observe stoichiometric and sub-stoichiometric cofactors, likely including protein isomerases. Collectively, comprehensive analysis of ER-associated protein biogenesis ex vivo reveals numerous mechanistic insights advancing beyond the study of isolated components.
2023-02-03 | PXD035475 | Pride
Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2α, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2α/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2α S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2α phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2α, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.
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Project description:Local mRNA translation in axons is critical for the spatial and temporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons, however how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER – ribosome interactions causes defects in axon morphology. Our results establish an important role for the axonal ER in dynamically localizing mRNA translation which is important for proper neuron development.
2024-04-29 | GSE262262 | GEO
Project description:In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a role for the ER in global protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression.
2011-12-20 | GSE31539 | GEO
Project description:The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) causes stress and induces the unfolded protein response (UPR) which is characterised in part by the transcriptional induction of genes involved in assisting protein folding. Translational responses to ER stress have been less well described and here we report on a genome-wide analysis of translational regulation in the response to the ER stress-inducing agent dithiothreitol (DTT) in Saccharomyces cerevisiae. Although the observed polysome profiles were similar under control and ER stress conditions microarray analysis identified transcipt-specific translational regulation. Genes with functions in ribosomal biogenesis and assembly were translationally repressed under ER stress. In contrast mRNAs for known UPR genes, including the UPR transcription factor HAC1, the ER-oxidoreductase ERO1 and the ER-associated protein degradation (ERAD) gene DER1 were enriched in polysomal fractions under ER stress conditions. In addition, we show that splicing of HAC1 mRNA is required for efficient ribosomal loading and that Gcn2p is required for normal HAC1 splicing, so shedding light on the role of this protein kinase in the UPR pathway. Experiment Overall Design: Polyribosomes were extracted from S. cerevisiae cells treated with 2 mM DTT or water (control), and fractionated according to ribosome loading. Following RNA purification from these fractions, for each sub-polysomal and polysomal RNA sample, fractions from three independent extracts per treatment (DTT/control) were pooled.
2008-06-11 | E-GEOD-10091 | biostudies-arrayexpress
Project description:eIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.
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Project description:Translational control during the intricate process of sporulation in Bacillus subtilis as a response to nutrient limitation is still underexplored. Here, we employed a comprehensive approach including RNA-seq, ribosome profiling and fluorescence microscopy to dissect the translational landscape of B. subtilis during sporulation. We identified two events of translation silencing and described the spatiotemporal changes in the subcellular location of translational machinery during sporulation. Using a triple knock-out strain (3KO) of zinc-independents paralogs of three zinc-dependent ribosomal proteins L31, L33 and S14, we investigated the potential regulatory role of ribosome during sporulation. The 3KO strain exhibited delayed sporulation, reduced germination efficiency, and dysregulated translation including expression of key metabolic and sporulation-related genes as well as disruptions in translation silencing, particularly in late sporulation.
2023-12-06 | GSE249449 | GEO