scholarly journals Genome-wide survey of ribosome collision

2019 ◽  
Author(s):  
Peixun Han ◽  
Mari Mito ◽  
Yuichi Shichino ◽  
Satoshi Hashimoto ◽  
Tsuyoshi Udagawa ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosome Profiling ◽  
Unfolded Protein ◽  
Genome Wide ◽  
A Genome ◽  
The Unfolded Protein Response ◽  
Ribosome Pausing ◽  
Protein Response ◽  
Insight Into ◽  
Genome Wide Survey

AbstractIn protein synthesis, ribosome movement is not always smooth and is rather often impeded for numerous reasons. Although the deceleration of the ribosome defines the fates of the mRNAs and synthesizing proteins, fundamental issues remain to be addressed, including where ribosomes pause in mRNAs, what kind of RNA/amino acid sequence causes this pause, and the physiological significance of this slowdown of protein synthesis. Here, we surveyed the positions of ribosome collisions caused by ribosome pausing in humans and zebrafish on a genome-wide level using modified ribosome profiling. The collided ribosomes, i.e., disomes, emerged at various sites: the proline-proline-lysine motif, stop codons, and the 3′ untranslated region (UTR). The number of ribosomes involved in a collision is not limited to two, but rather four to five ribosomes can form a queue of ribosomes. In particular, XBP1, a key modulator of the unfolded protein response, shows striking queues of collided ribosomes and thus acts as a substrate for ribosome-associated quality control (RQC) to avoid the accumulation of undesired proteins in the absence of stress. Our results provide an insight into the causes and consequences of ribosome slowdown by dissecting the specific architecture of ribosomes.

scholarly journals A genome-wide screen for ER autophagy highlights key roles of mitochondrial metabolism and ER-resident UFMylation

2019 ◽  
Author(s):  
Jin Rui Liang ◽  
Emily Lingeman ◽  
Thao Luong ◽  
Saba Ahmed ◽  
Truc Nguyen ◽  
...  
Keyword(s):  
Mitochondrial Metabolism ◽  
Unfolded Protein ◽  
Selective Degradation ◽  
Genome Wide ◽  
Energy Sensor ◽  
A Genome ◽  
Cellular Logic ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Insight Into

SummarySelective degradation of organelles via autophagy is critical for cellular differentiation, homeostasis, and organismal health. Autophagy of the ER (ER-phagy) is implicated in human neuropathy but is poorly understood beyond a few specialized autophagosomal receptors and remodelers. Using an ER-phagy reporter and genome-wide CRISPRi screening, we identified 200 high-confidence factors involved in human ER-phagy. We mechanistically investigated two pathways unexpectedly required for ER-phagy. First, reduced mitochondrial metabolism represses ER-phagy, which reverses the logic of general autophagy. Mitochondrial crosstalk with ER-phagy bypasses the energy sensor AMPK, instead directly impacting ULK1. Second, ER-localized UFMylation is required for ER-phagy that represses the unfolded protein response. The UFL1 ligase is brought to the ER surface by DDRGK1, analogous to PINK1-Parkin regulation during mitophagy. Our data provide insight into the unique cellular logic of ER-phagy, reveal parallels between organelle autophagies, and provide an entry point to the relatively unexplored process of degrading the ER network.

scholarly journals Regulated Ire1-dependent mRNA decay requires no-go mRNA degradation to maintain endoplasmic reticulum homeostasis in S. pombe

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Nicholas R Guydosh ◽  
Philipp Kimmig ◽  
Peter Walter ◽  
Rachel Green
Keyword(s):  
Endoplasmic Reticulum ◽  
Critical Role ◽  
Mrna Degradation ◽  
Ribosome Profiling ◽  
Unfolded Protein ◽  
Selective Degradation ◽  
Stressed Cells ◽  
Novel Target ◽  
The Unfolded Protein Response ◽  
Protein Response

The unfolded protein response (UPR) monitors and adjusts the protein folding capacity of the endoplasmic reticulum (ER). In S. pombe, the ER membrane-resident kinase/endoribonuclease Ire1 utilizes a mechanism of selective degradation of ER-bound mRNAs (RIDD) to maintain homeostasis. We used a genetic screen to identify factors critical to the Ire1-mediated UPR and found several proteins, Dom34, Hbs1 and Ski complex subunits, previously implicated in ribosome rescue and mRNA no-go-decay (NGD). Ribosome profiling in ER-stressed cells lacking these factors revealed that Ire1-mediated cleavage of ER-associated mRNAs results in ribosome stalling and mRNA degradation. Stalled ribosomes iteratively served as a ruler to template precise, regularly spaced upstream mRNA cleavage events. This clear signature uncovered hundreds of novel target mRNAs. Our results reveal that the UPR in S. pombe executes RIDD in an intricate interplay between Ire1, translation, and the NGD pathway, and establish a critical role for NGD in maintaining ER homeostasis.

scholarly journals Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress inSaccharomyces cerevisiae

2009 ◽  
Vol 20 (5) ◽  
pp. 1493-1508 ◽  
Author(s):  
Shi-Xiong Tan ◽  
Mariati Teo ◽  
Yuen T. Lam ◽  
Ian W. Dawes ◽  
Gabriel G. Perrone
Keyword(s):  
Endoplasmic Reticulum ◽  
Superoxide Dismutase ◽  
Er Stress ◽  
Stress Sensitivity ◽  
Pentose Phosphate ◽  
Unfolded Protein ◽  
Genome Wide ◽  
The Unfolded Protein Response ◽  
Protein Response

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.

scholarly journals Complementary Roles of GADD34- and CReP-Containing Eukaryotic Initiation Factor 2α Phosphatases during the Unfolded Protein Response

2016 ◽  
Vol 36 (13) ◽  
pp. 1868-1880 ◽  
Author(s):  
David W. Reid ◽  
Angeline S. L. Tay ◽  
Jeyapriya R. Sundaram ◽  
Irene C. J. Lee ◽  
Qiang Chen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Unfolded Protein Response ◽  
Control Cell ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Current View ◽  
Eukaryotic Initiation Factor ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation in stressed cells. While phosphorylated eIF2α (P-eIF2α) attenuates global protein synthesis, mRNAs encoding stress proteins are more efficiently translated. Two eIF2α phosphatases, containing GADD34 and CReP, catalyze P-eIF2α dephosphorylation. The current view of GADD34, whose transcription is stress induced, is that it functions in a feedback loop to resolve cell stress. In contrast, CReP, which is constitutively expressed, controls basal P-eIF2α levels in unstressed cells. Our studies show that GADD34 drives substantial changes in mRNA translation in unstressed cells, particularly targeting the secretome. Following activation of the unfolded protein response (UPR), rapid translation ofGADD34mRNA occurs and GADD34 is essential for UPR progression. In the absence of GADD34, eIF2α phosphorylation is persistently enhanced and the UPR translational program is significantly attenuated. This “stalled” UPR is relieved by the subsequent activation of compensatory mechanisms that include AKT-mediated suppression of PKR-like kinase (PERK) and increased expression ofCRePmRNA, partially restoring protein synthesis. Our studies highlight the coordinate regulation of UPR by the GADD34- and CReP-containing eIF2α phosphatases to control cell viability.

The unfolded protein response is triggered following a single, unaccustomed resistance-exercise bout

2014 ◽  
Vol 307 (6) ◽  
pp. R664-R669 ◽  
Author(s):  
Daniel I. Ogborn ◽  
Bryon R. McKay ◽  
Justin D. Crane ◽  
Gianni Parise ◽  
Mark A. Tarnopolsky
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Resistance Exercise ◽  
Unfolded Protein Response ◽  
Initiation Factor ◽  
Knee Extensors ◽  
Leg Extension ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress results from an imbalance between the abundance of synthesized proteins and the folding capacity of the ER. In response, the unfolded protein response (UPR) attempts to restore ER function by attenuating protein synthesis and inducing chaperone expression. Resistance exercise (RE) stimulates protein synthesis; however, a postexercise accumulation of unfolded proteins may activate the UPR. Aging may impair protein folding, and the accumulation of oxidized and misfolded proteins may stimulate the UPR at rest in aged muscle. Eighteen younger ( n = 9; 21 ± 3 yr) and older ( n = 9; 70 ± 4 yr) untrained men completed a single, unilateral bout of RE using the knee extensors (four sets of 10 repetitions at 75% of one repetition maximum on the leg press and leg extension) to determine whether the UPR is increased in resting, aged muscle and whether RE stimulates the UPR. Muscle biopsies were taken from the nonexercised and exercised vastus lateralis at 3, 24, and 48 h postexercise. Age did not affect any of the proteins and transcripts related to the UPR. Glucose-regulated protein 78 (GRP78) and protein kinase R-like ER protein kinase (PERK) proteins were increased at 48 h postexercise, whereas inositol-requiring enzyme 1 alpha (IRE1α) was elevated at 24 h and 48 h. Despite elevated protein, GRP78 and PERK mRNA was unchanged; however, IRE1α mRNA was increased at 24 h postexercise. Activating transcription factor 6 (ATF6) mRNA increased at 24 h and 48 h, whereas ATF4, CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage protein 34 mRNA were unchanged. These data suggest that RE activates specific pathways of the UPR (ATF6/IRE1α), whereas PERK/eukaryotic initiation factor 2 alpha/CHOP does not. In conclusion, acute RE results in UPR activation, irrespective of age.

scholarly journals Manipulation of the unfolded protein response: A pharmacological strategy against coronavirus infection

2021 ◽  
Vol 17 (6) ◽  
pp. e1009644
Author(s):  
Liliana Echavarría-Consuegra ◽  
Georgia M. Cook ◽  
Idoia Busnadiego ◽  
Charlotte Lefèvre ◽  
Sarah Keep ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Hepatitis Virus ◽  
Ribosome Profiling ◽  
Murine Hepatitis Virus ◽  
Unfolded Protein ◽  
Virion Release ◽  
Infected Cells ◽  
Coronavirus Infection ◽  
The Unfolded Protein Response ◽  
Protein Response

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

scholarly journals Putting the brakes on the unfolded protein response

2011 ◽  
Vol 193 (1) ◽  
pp. 17-19 ◽  
Author(s):  
Frank Sicheri ◽  
Robert H. Silverman
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Unfolded Protein Response ◽  
Kinase Domain ◽  
Unfolded Protein ◽  
Cell Biol ◽  
Switch Mechanism ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Insight Into

The unfolded protein response is an ancient cellular pathway for rapidly responding to endoplasmic reticulum stress. Two studies in this issue (Rubio et al. 2011. J. Cell. Biol. doi:10.1083/jcb.201007077 and Chawla et al. 2011. J. Cell. Biol. doi:10.1083/jcb.201008071) provide insight into how the unfolded protein response is tamped down to restore normal endoplasmic reticulum function. Although both papers implicate the Ire1 kinase domain as the key effector of the off-switch mechanism, alternate models for how this is achieved are proposed.

scholarly journals Using targeted genome integration for virus-free genome-wide mammalian CRISPR screen

2020 ◽  
Author(s):  
Kai Xiong ◽  
Karen Julie la Cour Karottki ◽  
Hooman Hefzi ◽  
Songyuan Li ◽  
Lise Marie Grav ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
Clonal Variation ◽  
Unfolded Protein ◽  
Level Ii ◽  
Genome Wide ◽  
Crispr Screen ◽  
Screening Platform ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACTPooled CRISPR screens have been widely applied in mammalian cells to discover genes regulating various phenotypes of interest. In such screens, CRISPR components are generally delivered with a lentivirus. However, lentiviral CRISPR screens are limited by unpredictable genome insertion, the requirement of biosafety level II lab facilities and personnel trained to work with viruses. Here we established a virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 74,617 gRNAs targeting 18,353 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad thus reducing the clonal variation. Using this VF CRISPR screening platform, 338 genes are identified as essential for CHO cell growth and 76 genes were found to be involved in the unfolded protein response (UPR) induced endoplasmic reticulum (ER) stress. Extensive validation of the candidate genes further demonstrated the robustness of this novel non-viral CRISPR screen method.

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