Regulation of translation under mitoprotein-induced stress in Saccharomyces cerevisiae
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE172017 | GEO | 2021/12/14
REPOSITORIES: GEO
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Project description:Mitochondrial functions are largely performed by proteins imported from cytosol. Impaired protein import in mitochondria affects the maintenance of intra-mitochondrial anabolic and energy metabolic pathways. It leads to cytosolic accumulation of mistargeted proteins and activation of cellular stress responses. To explore the communication between these mechanism in mammalian system, we targeted the co-chaperone of mitochondrial Hsp70, a GrpE Like protein 1 (Grpel1), in mice. We show that loss of protein import into mitochondrial matrix results in mitochondrial dysfunction, which triggers stress responses. The total shut down of mtHSP70 function by overall knockout of Grpel1 resulted in early embryonic lethality, and tamoxifen-induced skeletal muscle-specific knockout of Grpel1 caused rapid muscular atrophy. We show here, with transcriptomics analysis, that protein-import failure in mitochondria increased cellular proteotoxic stress due to mitochondrial dysfunction. As a control mechanism, it triggered adaptive stress responses, including unfolded protein responses and integrated stress responses. Metabolic profiling of skeletal muscle-specific knockout mice revealed amino acid and TCA cycle intermediates shuttle between serum and muscle.
2022-10-04 | GSE198540 | GEO
Project description:The potency and indiscriminate nature of formaldehyde reactivity upon biological molecules make it a universal stressor. However, some organisms such as Methylorubrum extorquens possess means to rapidly and effectively mitigate formaldehyde-induced damage. EfgA is a recently identified formaldehyde sensor predicted to halt translation in response to elevated formaldehyde as a means to protect cells. Herein, we investigate growth, formaldehyde consumption, and changes in gene expression to better understand how M. extorquens responds to formaldehyde with and without the EfgA-formaldehyde-mediated translational response, and how this mechanism compares to other forms of translation inhibition. These distinct mechanisms of translation inhibition have notable differences: they each involve different specific players and in addition, formaldehyde also acts as a general, multi-target stressor and a potential carbon source. Here, we present findings demonstrating that in addition to its characterized impact on translation, functional EfgA also allows for a rapid and robust transcriptional response to formaldehyde and that removal of efgA leads to heightened proteotoxic and genotoxic stress in the presence of increased formaldehyde levels. As our previous work suggested that formaldehyde is proteotoxic in M. extorquens, we accurately predicted that strains lacking efgA would experience increased protein damage. We also found that many downstream consequences of translation inhibition were shared by EfgA-formaldehyde- and kanamycin-mediated translation inhibition. Our work to modularize the transcriptional response uncovered additional layers of regulatory control enacted by functional EfgA upon experiencing formaldehyde stress, and further demonstrate the importance this protein plays at both transcriptional and translational levels in this model methylotroph.
2020-12-31 | GSE163955 | GEO
Project description:Current evidence suggests that nuclear-encoded mitochondrial proteins can be locally translated at the mitochondrial surface and co-translationally or post-translationally imported into mitochondria. mRNA localization on the mitochondrial membrane, a prerequisite for localized translation, remains uncharacterized in higher eukaryotic organisms. We employed fractionation-sequencing to profile mitochondria-associated mRNAs in zebrafish larvae. Our transcriptome-wide analysis reveals the localization of mRNAs of only 12% of the nuclear-encoded mitochondrial proteins to the mitochondrial surface, which suggests that post-translational import is the dominant mode of protein import to mitochondria. Additionally, the mRNAs which were localized to the mitochondrial membrane consisted mostly of those encoding proteins involved in mitochondrial dynamics, suggesting their site-specific translation. Finally, we show that the loss of function of the MIA pathway responsible for the post-translational import of a subclass of mitochondrial proteins, triggers mitochondrial localization of mRNAs encoding proteins that are imported to mitochondria via other pathways. Thus, our study suggests that mRNA targeting and localized translation could be relevant in higher eukaryotes to combat stress conditions affecting mitochondrial biogenesis in general.
2023-10-28 | GSE160355 | GEO
Project description:Most mitochondrial proteins are encoded in the nucleus and require mitochondrial import after translation in the cytosol. A number of mutations in the mitochondrial protein import machinery have been shown to lead to human pathologies. However, a lack suitable tools to measure mitochondrial protein uptake has prevented determination of import rates across the mitochondrial proteome and identification of specific proteins affected by perturbation. Here, we introduce a pulsed-SILAC based proteomics approach that includes a booster signal to selectively increase the sensitivity for mitochondrial proteins, enabling dynamic analysis of mitochondrial protein uptake at the global scale. We applied this method to determine protein uptake kinetics and examined how inhibitors of different mitochondrial import machineries affect protein uptake in sub-mitochondrial compartments. Monitoring changes in translation and protein uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated the import- and translation machineries, via activation of the integrated stress response. Strikingly, uptake changes were not uniform with three groups of protein identified that showed no changes in uptake, or changes driven by reduced translation or import capacity. This study provides with a quantitative proteomics method to monitor mitochondrial protein uptake at a global scale to provide with insight into uptake rearrangements upon perturbation.
2021-12-18 | PXD022521 | Pride
Project description:Most mitochondrial proteins are encoded in the nucleus and require mitochondrial import after translation in the cytosol. A number of mutations in the mitochondrial protein import machinery have been shown to lead to human pathologies. However, a lack suitable tools to measure mitochondrial protein uptake has prevented determination of import rates across the mitochondrial proteome and identification of specific proteins affected by perturbation. Here, we introduce a pulsed-SILAC based proteomics approach that includes a booster signal to selectively increase the sensitivity for mitochondrial proteins, enabling dynamic analysis of mitochondrial protein uptake at the global scale. We applied this method to determine protein uptake kinetics and examined how inhibitors of different mitochondrial import machineries affect protein uptake in sub-mitochondrial compartments. Monitoring changes in translation and protein uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated the import- and translation machineries, via activation of the integrated stress response. Strikingly, uptake changes were not uniform with three groups of protein identified that showed no changes in uptake, or changes driven by reduced translation or import capacity. This study provides with a quantitative proteomics method to monitor mitochondrial protein uptake at a global scale to provide with insight into uptake rearrangements upon perturbation.
2021-12-18 | PXD024054 | Pride
Project description:Most mitochondrial proteins are encoded in the nucleus and require mitochondrial import after translation in the cytosol. A number of mutations in the mitochondrial protein import machinery have been shown to lead to human pathologies. However, a lack suitable tools to measure mitochondrial protein uptake has prevented determination of import rates across the mitochondrial proteome and identification of specific proteins affected by perturbation. Here, we introduce a pulsed-SILAC based proteomics approach that includes a booster signal to selectively increase the sensitivity for mitochondrial proteins, enabling dynamic analysis of mitochondrial protein uptake at the global scale. We applied this method to determine protein uptake kinetics and examined how inhibitors of different mitochondrial import machineries affect protein uptake in sub-mitochondrial compartments. Monitoring changes in translation and protein uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated the import- and translation machineries, via activation of the integrated stress response. Strikingly, uptake changes were not uniform with three groups of protein identified that showed no changes in uptake, or changes driven by reduced translation or import capacity. This study provides with a quantitative proteomics method to monitor mitochondrial protein uptake at a global scale to provide with insight into uptake rearrangements upon perturbation.
2021-12-20 | PXD024066 | Pride
Project description:We investigated genome-wide changes in mRNA translation in Arabidopsis thaliana suspension cell cultures exposed to brief perids of two types of stress: elevated temperature (37 degree_C) and high salinity (200 mM NaCl). To this end, we subjected polysomal RNA and non-polysomal RNA from sucrose gradient fractionated cell lysates to the co-hybridization on Agilent Arabidopsis 3 Oligo Microarrays. The ratio of signal intensities (polysomal RNA: non-polysomal RNA) was used as an indicator of the translation state for each transcript. To inspect coordination of changes in translational profiles with transcriptional profiles, we also isolated total RNAs from the same cells used for translational profiling experiments and investigated changes in accumulated transcript levels in response to each stress using the microarray. Two biological replicates were analyzed.
2010-07-07 | E-DORD-69 | biostudies-arrayexpress
Project description:The unfolded protein response (UPR) couples cellular translation rates and gene expression to the protein folding status of the endoplasmic reticulum (ER). Upon activation, the UPR machinery elicits a general suppression of protein synthesis and activation of stress gene expression, which act coordinately to restore protein folding homeostasis. We report here that UPR activation promotes the release of signal sequence-encoding mRNAs from the ER to the cytosol as a mechanism to decrease protein influx into the ER. This release of mRNA begins rapidly, then gradually recovers with ongoing stress. Upon release into the cytosol, these mRNAs have divergent fates: some synthesize full-length proteins, while others are translationally inactive and retain nascent protein chains. Together, these findings identify the dynamic subcellular localization of mRNAs and translation as a regulatory feature of the cellular response to protein folding stress. Cells were treated with a timecourse of Thapsigargin or DTT, then fractionated and analyzed by mRNA-seq or ribosome profiling
2014-08-28 | E-GEOD-53743 | biostudies-arrayexpress
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.
2016-07-03 | E-GEOD-68265 | biostudies-arrayexpress
Project description:Co-Translational Response to Proteotoxic Stress by Elongation Pausing of Ribosomes
| PRJNA181016 | ENA