Transcriptome partitioning for mRNA translation in hypoxia
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE49029 | GEO | 2015/04/01
SECONDARY ACCESSION(S): PRJNA212707
REPOSITORIES: GEO
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Project description:Protein synthesis belongs to the most energy consuming processes in the cell. Lowering oxygen tension below normal (hypoxia) causes a rapid inhibition of global mRNA translation due to the decreased availability of energy. Interestingly, subsets of mRNAs pursue active translation under such circumstances. In human fibrosarcoma cells (HT1080) exposed to prolonged hypoxia (36 h, 1% oxygen) we observed that transcripts are either increasingly or decreasingly associated with ribosomes localized at the endoplasmic reticulum (ER). In a global setting it turned out that only 31% of transcripts showing elevated total-RNA levels were also increasingly present at the ER in hypoxia. These genes, regulated by its expression as well as its ER-localization, belong to the gene ontology’s “hypoxia response”, “glycolysis” and “HIF-1 transcription factor network” supporting the view of active mRNA translation at the ER during hypoxia. Interestingly, a large group of RNAs was found to be unchanged at the expression level, but translocate to the ER in hypoxia. Among these are transcripts encoding translation factors and >180 ncRNAs. In summary, we provide evidence that protein synthesis is favoured at the ER and, thus, partitioning of the transcriptome between cytoplasmic and ER associated ribosomes mediates adaptation of gene expression in hypoxia. Human fibrosarcoma HT1080 cells were cultured for 36 h under control/atmospheric (21% O2, 5% CO2, 37 °C) or hypoxic (1% O2, 5% CO2, 37°C) conditions. Total RNA (representing expression level) and ER (endoplasmatic reticulum)-RNA (representing transcript localization at ER) was isolated for both conditions. Pooled RNA samples from 5 independent biological experiments were used for microarray analysis.
2015-04-01 | E-GEOD-49029 | biostudies-arrayexpress
Project description:Transcripts encoding membrane and secreted proteins are known to have translation on endoplasmic reticulum (ER) through the signal recognition particle (SRP)-mediated mechanism. Translation-independent association of mRNAs with ER has also been reported, but poorly understood. Here, using 3’ end sequencing, we examine transcripts associated with polysomes on ER in mouse C2C12 cells. We identify transcript features that facilitate translation-independent ER association, including CDS size, 3’UTR size, and GC content. Interestingly, transcripts encoding nuclear proteins tend to have a higher ER association potential than other transcripts, suggesting localized translation to facilitate nuclear import. We show long 3’UTR isoforms are generally more likely to be associated with ER than short isoforms. Consistently, global shortening of 3’UTRs by knocking down CFI-25 expression leads to substantial alternation of mRNA association with ER. While ER association potentials of transcripts undifferentiated myoblasts are similar to those in differentiated myotubes, 3’UTR lengthening in differentiation leads to more transcripts associated with ER in myotubes. Moreover, translation-independent ER association correlates with low mRNA stability, which is exacerbated in cells with oxidative stress. Together, our data indicate that sequence and structure features of mRNAs facilitate ER association and translation, potentially impacting localization dynamics of protein products, and cellular stress remodels membrane-associated transcripts through mRNA stability regulation.
2023-04-15 | GSE150607 | GEO
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.
2014-06-02 | E-GEOD-54581 | biostudies-arrayexpress
Project description:Background Protein synthesis is a highly energy demanding process and is regulated according to energy availability in plant cells. Light and sugar availability affect mRNA translation but the specific roles of these factors remain unclear. In this study, sucrose was applied to Arabidopsis seedlings kept in the light or in the dark, in order to distinguish sucrose and light effects on transcription and translation. These were studied using microarray analysis of steady state mRNA and mRNA bound to translating ribosomes. Results Steady state mRNA levels were affected differently by sucrose in the light and in the dark but general translation increased to a similar extend in both conditions. Alterations in polysomal mRNA association closely followed the changes induced on the transcript level. However, for 243 mRNAs, a change in ribosome occupancy was observed after sucrose treatment in the light, but not in the dark condition. Many of these mRNAs are annotated as encoding ribosomal proteins, supporting specific translational regulation of this group of transcripts. Unexpectedly, the numbers of ribosomes bound to each mRNA decreased for mRNAs with increased ribosome occupancy. Conclusions Our results suggest that sucrose regulate translation of these 243 mRNAs but specifically in the light, through a novel regulatory mechanism. Our data sows that increased polysomal association is not necessarily leading to more ribosomes per transcript, suggesting a mechanism of translational induction not solely dependent on increased translation initiation rates. Four different samples groups were used: Control, Darkness, Sucrose, Simultaneous Darkness and sucrose treatments. All sample groups was represented by both total mRNA hybridization and of mRNAs from polysomal enrichment. All sample groups were represented by three biological replicates.
2015-01-28 | E-GEOD-59306 | biostudies-arrayexpress
Project description:The eukaryotic translation initiation factor 5B (eIF5B) is a homolog of the ancient IF2, a translation factor that facilitates initiator methionine-tRNAiMet (met-tRNAiMet) delivery to ribosomes in prokaryotes. IF2 evolved during early anaerobic cellular life and can be traced back to the last universal common ancestor. Here, we show that eIF5B is essential for eukaryotic hypoxia tolerance, reminiscent of how IF2 supports prokaryotic anaerobiosis. Global protein synthesis analyses identified eIF5B as a hypoxia-specific translation factor involved in translocating met-tRNAiMet to initiating ribosomes. Systemic translatome studies revealed that eIF5B-dependent mRNAs encode proteins of central carbon metabolism, fructolysis, and other pathways enhanced in organisms inhabiting hypoxic niches. These pathways rely preferentially on eIF5B rather than eIF2, the canonical initiation factor that delivers met-tRNAiMet during aerobic eukaryotic protein synthesis. We suggest that eIF5B/IF2 was retained during evolution of the aerobic eukaryotic lineage to cope with episodes of oxygen deprivation.
2018-01-03 | PXD006799 | Pride
Project description:Oxygen and glucose metabolism plays a pivotal role in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) occurs during ischemia and stroke, resulting in extensive tissue injury and cell death. We applied time-resolved ribosome profiling technique to assess early events at the level of gene expression in rat pheochromocytoma PC12 cells during short-term OGD. Most substantial alterations in transcripts levels and their translation were seen to occur in the first 20 minutes of OGD. The rapid adaptation of translation apparatus to OGD is global and involves altered elongation and initiation rates. We also observed salient and reproducible alterations in ribosome densities of individual mRNAs such as increased translation of particular upstream Open Reading Frames (uORFs); induced site-specific arrests of the ribosomes and synthesis of extended protein isoforms. Ribosome profiling (with mRNA-seq sequencing) was carried out at 0,20,40 and 60 minutes of OGD. Two biological replicates were used.
2015-04-10 | E-GEOD-60752 | biostudies-arrayexpress
Project description:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.
2016-08-03 | E-GEOD-74393 | biostudies-arrayexpress
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:Low oxygen stress dynamically regulates the translation of cellular mRNAs as a means of energy conservation in seedlings of Arabidopsis thaliana. Most of the highly hypoxia-induced mRNAs are recruited to polysomes and actively translated, whereas other cellular mRNAs become translationally inactive and are either targeted for stabilization or degradation. Here we identify the involvement of OLIGOURIDYLATE BINDING PROTEIN 1 (UBP1), a triple RNA Recognition Motif protein, in dynamic and reversible aggregation of translationally repressed mRNAs during hypoxia. Mutation or downregulation of UBP1C interferes with seedling establishment and reduces survival of low oxygen stress. By use of messenger ribonucleoprotein immunopurification, we show that UBP1C constitutively binds a subpopulation of mRNAs characterized by U-rich 3M-bM-^@M-^Y-untranslated regions under normoxic conditions. During hypoxia, UBP1C association with non-U-rich mRNAs is enhanced concomitant with its aggregation into microscopically visible cytoplasmic foci, referred to as UBP1 stress granules (SGs). This UBP1C-mRNA association occurs as global levels of protein synthesis decline. Upon reoxygenation, rapid UBP1 SG disaggregation coincides with the return of the stabilized mRNAs to polysomes. The mRNAs that are highly induced and translated during hypoxia largely circumvent UBP1C sequestration. Thus, UBP1 is established as a component of dynamically assembled cytoplasmic mRNPs that sequester mRNAs that are poorly translated during a transient low energy stress. Immunoprecipated RNA associated with Arabidopsis UBP1C (IP) was compared with total cellular RNA from light (L), mock dark (D), 2 h hypoxia, and 2 h hypoxia + 20 min reoxygenation treated samples with duplicate hybridizations to the Affymetrix ATH1 Genechip array.
2014-01-13 | E-GEOD-49411 | biostudies-arrayexpress
Project description:We investigated the association of cryptic unstable transcripts (CUTs) to ribosomes in Saccharomyces cerevisiae. We used 5’cap-sequence followed by sucrose gradient fractionation of polyribosome fractions after ultracentrifugation to assess the relative association of transcripts to the ribosomes. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We performed 5PSeq after glucose depletion to increase the number of ribosomes stop at the 5’UTR and start codon.
2019-10-01 | GSE119134 | GEO