A functional link between the co-translational protein translocation pathway and the UPR

Plumb, Rachel; Zhang, Zai-Rong; Appathurai, Suhila; Mariappan, Malaiyalam

doi:10.7554/elife.07426

Cited by 85 publications

(139 citation statements)

References 50 publications

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“…Collectively, we concluded that XBP1u mRNA is specifically recruited to the ER membrane by the SRP-mediated ERtargeting pathway. Our findings regarding SRP involvement in the ER-specific targeting of XBP1u are consistent with previous findings based on different approaches, although the role of translational pausing of XBP1u in the recognition of XBP1u by SRP was not previously evaluated (34).…”

Section: Discussionsupporting

confidence: 82%

“…Because an N-terminal signal sequence causes cotranslational translocation of the protein into the luminal space of the ER, the mRNA should remain on the translocon, which indicates that the mRNA is able to be spliced on the translocon with high efficiency. This notion is consistent with a recently published paper showing that IRE1α directly associates with a translocon that could splice XBP1u mRNA (34).…”

Section: Discussionsupporting

confidence: 82%

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Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway

Kanda

Yanagitani

Yokota

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Unconventional mRNA splicing on the endoplasmic reticulum (ER) membrane is the sole conserved mechanism in eukaryotes to transmit information regarding misfolded protein accumulation to the nucleus to activate the stress response. In metazoans, the unspliced form of X-box-binding protein 1 (XBP1u) mRNA is recruited to membranes as a ribosome nascent chain (RNC) complex for efficient splicing. We previously reported that both hydrophobic (HR2) and translational pausing regions of XBP1u are important for the recruitment of its own mRNA to membranes. However, its precise location and the molecular mechanism of translocation are unclear. We show that XBP1u-RNC is specifically recruited to the ER membrane in an HR2-and translational pausing-dependent manner by immunostaining, fluorescent recovery after photobleaching, and biochemical analyses. Notably, translational pausing during XBP1u synthesis is indispensable for the recognition of HR2 by the signal recognition particle (SRP), resulting in efficient ER-specific targeting of the complex, similar to secretory protein targeting to the ER. On the ER, the XBP1u nascent chain is transferred from the SRP to the translocon; however, it cannot pass through the translocon or insert into the membrane. Therefore, our results support a noncanonical mechanism by which mRNA substrates are recruited to the ER for unconventional splicing. and subsequent protein folding are associated with a number of diseases. Accordingly, a detailed understanding of the mechanisms that mediate these complex processes is necessary. In eukaryotes, secretory proteins are initially translated by cytosolic ribosomes. When the N-terminal signal peptide reaches outside of the peptide exit channel in the ribosome, the ribosome nascent chain (RNC) complex is recognized by the signal recognition particle (SRP) and peptide elongation is slowed (1-5). This SRP-RNC complex is then recruited to the ER via the affinity between SRP and SRP receptor (SR) that exists in the ER membrane (2, 3). The RNC complex is then delivered to the polypeptide channel in the ER (i.e., the Sec61 translocon) (6). SRP is released from the RNC, which cancels the slowed-down elongation. As a result, the ER-targeted ribosome cotranslationally translocates its synthesizing polypeptide into the luminal space of the ER. The translocated protein is folded into its native 3D structure with the help of molecular chaperones and folding enzymes (7). The folded proteins are sorted to their final destinations to exert their functions.The burden of new proteins entering the ER varies widely among conditions, such as cell differentiation, environmental conditions, and the physiological state of the cell (8, 9). In addition, the folding capacity in the ER is easily compromised by many stressful conditions, including glucose starvation, virus infection, and perturbations in the Ca 2+ concentration. Therefore, it is necessary for cells to manage the imbalance between the load of newly entering proteins and the folding capacity in the ER, and this i...

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Section: Discussionsupporting

confidence: 82%

Section: Discussionsupporting

confidence: 82%

Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway

Kanda

Yanagitani

Yokota

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Perhaps the most intriguing example of branch specificity was our observation that depletion of translocon subunits led to selective activation of the IRE1α branch, which is notable in light of recent studies suggesting that IRE1α, unlike ATF6 or PERK, acts in physical association with the translocon (Plumb et al, 2015). Given that we, in agreement with others (Shoulders et al, 2013), observed regulation of translocon expression to be uniquely under IRE1α control, this suggests a feedback model in which IRE1α monitors the state of translocation.…”

Section: Discussionmentioning

confidence: 58%

“…Three out of four subunits of the translocon-associated protein complex (TRAP) scored; and strikingly, among the 7 hits with the strongest phenotypes were all three genes that encode the ER protein-translocation channel or translocon ( SEC61A1 , SEC61B , SEC61G ) (Figures 4D, 4E, S4D). The phenotypes of SRP-targeting factors and the translocon were surprising because recent reports have shown that SRP-mediated recruitment of unspliced XBP1 (XBP1u) to the ER and IRE1α binding to the translocon are required for maximal XBP1 splicing in response to exogenous stress (Kanda et al, 2016; Plumb et al, 2015). Satisfyingly, targeting of both ERN1 (IRE1α) and XBP1 decreased reporter signal in the screen (Figure 4D).…”

Section: Resultsmentioning

confidence: 99%

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Adamson

Norman

Jost

et al. 2016

Cell

896

947

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SUMMARY Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ~100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

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“…Regulated IRE1-dependent decay of mRNA, a key component of the unfolded protein response, results in degradation of viral mRNA transcripts and spliced Xbp1 (13,35). However, there are multiple lines of evidence suggesting that IRF3 activation can result from other forms of noninfectious cell damage that involve Xbp1 splicing (17,18,36).…”

Section: Discussionmentioning

confidence: 97%

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes

Iracheta-Vellve

Petrášek

Gyöngyösi

et al. 2016

Journal of Biological Chemistry

128

119

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Edited by Jeffrey PessinFibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl 4 administration to WT and IRF3-, Toll/ Interleukin-1R (TIR) domain-containing adapter-inducing interferon-␤ (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl 4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl 4 . Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl 4 . In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the proapoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

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A functional link between the co-translational protein translocation pathway and the UPR

Cited by 85 publications

References 50 publications

Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway

Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes

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