Regulated IRE1-dependent Decay Research Articles

Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

To relieve endoplasmic reticulum (ER) stress, IRE1 splices XBP1 messenger RNA (mRNA) or engages regulated IRE1-dependent decay (RIDD) of other mRNAs. Upon XBP1 deficiency, IRE1 switches to perform RIDD. We examined IRE1 in XBP1-deficient B cells and discovered that IRE1 undergoes phosphorylation at S729. We generated an anti-phospho-S729 antibody to investigate such phosphorylation. Compared with pharmacological ER stress inducers or Toll-like receptor ligands, the bacterial subtilase cytotoxin has an unusual capability in causing rapid and strong phosphorylation at S729 and triggering B cells to express spliced XBP1. To assess the function of S729 in IRE1, we generated S729A knock-in mice and found S729 is critically important for lipopolysaccharide-stimulated plasmablasts to respond to additional ER stress and for antibody production in response to immunization. We further crossed mice carrying an S729A mutation or ΔIRE1 (missing the kinase domain) with B cell-specific XBP1-deficient mice to trigger RIDD and discovered a critical role for S729 in regulating RIDD in B cells.

Dual IRE1 RNase functions dictate glioblastoma development.

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1‐dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1‐dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish.

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish.

Abstract LB-336: Specific serine phosphorylation of IRE-1 controls enhanced splicing of XBP-1 and regulated IRE-1-dependent decay (RIDD)

Abstract The endoplasmic reticulum (ER) stress sensor IRE-1 is critical for cancer survival and anti-tumor immunity. IRE-1 splices the mRNA of XBP-1 to activate the functional XBP-1s transcription factor or engages regulated IRE-1-dependent decay (RIDD) to degrade tissue-specific mRNAs. It is unclear how IRE-1 RNase activity is regulated to perform the two functions. Upon XBP-1 deficiency, IRE-1 switches to perform RIDD. We examined IRE-1 in XBP-1-deficient B cells, and discovered that IRE-1 undergoes glycosylation at asparagine 178 (N178) and phosphorylation at serine 729 (S729). We generated an anti-phospho-S729 antibody and confirmed that S729 is indeed phosphorylated in XBP-1-deficient B cells. Different from conventional ER stress inducers or Toll-like receptor ligands, subtilase cytotoxin (SubAB), an AB5 toxin produced by Shiga-toxigenic E. coli, has an unusual capability in causing rapid and strong phosphorylation of S729. To further understand the function of N178 and S729 of IRE-1, we generated two knock-in mouse models (N178Q and S729A). Compared with wild-type B cells and those carrying the N178Q mutation, B cells carrying the S729A mutation similarly respond to cues of differentiation by expressing XBP-1s, but they completely fail to respond to pharmacological ER stress inducers or SubAB, suggesting that phosphorylation of S729 is critically important for secondary ER stress response. To evaluate the role of N178, S729 and the kinase domain of IRE-1 in regulating RIDD, we crossed N178Q, S729A and ΔIRE-1 (deletion of amino acids 652 to 751 in the kinase domain) mice with B cell-specific XBP-1KO (CD19Cre/XBP-1f/f) mice to induce RIDD. RIDD was evidenced by the decreased mRNA levels of secretory immunoglobulin (Ig) μ heavy chains in XBP-1-deficient B cells. RIDD was blocked in S729A/XBP-1KO and ΔIRE-1/XBP-1KO but not in N178Q/XBP-1KO B cells. While deleting the kinase function of IRE-1 blocks both XBP-1 splicing and RIDD, mutating S729 only blocks RIDD, highlighting an important role of S729 in regulating RIDD. Since SubAB can efficiently trigger phosphorylation of S729 of IRE-1, we further demonstrated that exposure to SubAB indeed causes RIDD of secretory Ig μ heavy chains in wild-type B cells but not in those carrying the S729A mutation, further supporting the role of S729 in regulating RIDD. In addition, we showed that a potent inhibitor of the IRE-1 RNase, B-I09, can inhibit RIDD. The identification of phosphorylated S729 as a control for RIDD may help scientists devise new ways to specifically target this phosphorylation, contributing to the control of human cancer. Citation Format: Chih-Hang Tang, Shiun Chang, Adrienne Paton, James Paton, Dmitry Gabrilovich, Hidde Ploegh, Juan Del Valle, Chih-Chi Andrew Hu. Specific serine phosphorylation of IRE-1 controls enhanced splicing of XBP-1 and regulated IRE-1-dependent decay (RIDD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-336. doi:10.1158/1538-7445.AM2017-LB-336

Endoplasmic reticulum stress in the regulation of liver diseases: Involvement of Regulated IRE1α and β-dependent decay and miRNA.

Compromised protein folding capacity in the endoplasmic reticulum (ER) leads to a protein traffic jam that produces a toxic environment called ER stress. However, the ER smartly handles such a critical situation by activating a cascade of proteins responsible for sensing and responding to the noxious stimuli of accumulated proteins. The ER protein load is higher in secretory cells, such as liver hepatocytes, which are thus prone to stress-mediated toxicity and various diseases, including alcohol-induced liver injury, fatty liver disease, and viral hepatitis. Therefore, we discuss the molecular cues that connect ER stress to hepatic diseases. Moreover, we review the literature on ER stress-regulated miRNA in the pathogenesis of liver diseases to give a comprehensive overview of mechanistic insights connecting ER stress and miRNA in the context of liver diseases. We also discuss currently discovered regulated IRE1 dependent decay in regulation of hepatic diseases.

Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression.

Endoplasmic reticulum (ER) stress is implicated in many chronic diseases, but very little is known about how the unfolded protein response (UPR) responds to persistent ER stress in vivo. Here, we experimentally reconstituted chronic ER stress in the mouse liver, using repeated injection of a low dose of the ER stressor tunicamycin. Paradoxically, this treatment led to feedback-mediated suppression of a select group of mRNAs, including those encoding the ER chaperones BiP and GRP94. This suppression was due to both silencing of the ATF6α pathway of UPR-dependent transcription and enhancement of mRNA degradation, possibly via regulated IRE1-dependent decay (RIDD). The suppression of mRNA encoding BiP was phenocopied by ectopic overexpression of BiP protein, and was also observed in obese mice. Our findings suggest that persistent cycles of UPR activation and deactivation create an altered, quasi-stable setpoint for UPR-dependent transcriptional regulation-an outcome that could be relevant to conditions such as metabolic syndrome.

PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response.

The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). UPR can facilitate the restoration of cellular homeostasis, via the concerted activation of three ER stress sensors, namely IRE1, PERK and ATF6. Global approaches in several cellular contexts have revealed that UPR regulates the expression of many miRNAs that play an important role in the regulation of life and death decisions during UPR. Here we show that expression of miR-424(322)-503 cluster is downregulated during UPR. IRE1 inhibitor (4 μ8C) and deficiency of XBP1 had no effect on downregulation of miR-424(322)-503 during UPR. Treatment of cells with CCT030312, a selective activator of EIF2AK3/PERK signalling, leads to the downregulation of miR-424(322)-503 expression. The repression of miR-424(322)-503 cluster during conditions of ER stress is compromised in PERK-deficient MEFs. miR-424 regulates the expression of ATF6 via a miR-424 binding site in its 3′ UTR and attenuates the ATF6 transcriptional activity during UPR. Further miR-424 had no effect on IRE1-XBP1 axis but enhanced the regulated IRE1-dependent decay (RIDD). Our results suggest that miR-424 constitutes an obligatory fine-tuning mechanism where PERK-mediated downregulation of miR-424(322)-503 cluster regulates optimal activation of IRE1 and ATF6 during conditions of ER stress.

PDIA6 regulates insulin secretion by selectively inhibiting the RIDD activity of IRE1.

Protein disulfide isomerase A6 (PDIA6) interacts with protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme (IRE)-1 and inhibits their unfolded protein response signaling. In this study, shRNA silencing of PDIA6 expression in insulin-producing mouse cells reduced insulin production (5-fold) and, consequently, glucose-stimulated insulin secretion (3-4-fold). This inhibition of insulin release was independent of the PDIA6-PERK interaction or PERK activity. Acute inhibition of PERK did not change the short-term response of β cells to glucose. Rather, PDIA6 affected insulin secretion by modulating one of the activities of IRE1. At 11 mM glucose and lower, the regulated IRE1-dependent decay (RIDD) of the mRNA activity of IRE1 was activated, but not its X-box binding protein (XBP)-1 splicing activity. In the absence of PDIA6, RIDD activity toward insulin transcripts was enhanced up to 4-fold, as shown by molecular assays in cultured cells and the use of a fluorescent reporter in intact islets. Such physiologic activation of IRE1 by glucose contrasted with IRE1 activation by chemical stress, when both IRE1 activities were induced. Thus, whereas the stimulus determines the quality of IRE1 signaling, PDIA6 attenuates multiple enzymatic activities of IRE1, maintaining its signaling within a physiologically tolerable range.

Abstract 1253: Selectivity of regulated IRE1 dependent decay of RNA controls pro-oncogenic signaling in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most lethal form of glioma with an overall survival at 5 years nearly null (< 5%). Increasing evidences point towards the RNase activity of IRE1 as a central player in GBM development, particularly in cancer cell invasion and tumor vascularization. Indeed, IRE1 promotes XBP1 mRNA splicing, a well-described cytoprotective transcription factor that is required for cancer cell survival. Moreover, regulated IRE1 dependent decay of RNA (RIDD) leads to opposite effects on cell fate depending the ER stress intensity or duration and on the target RIDD substrates. Recent studies unraveled the presence of somatic mutations on the IRE1 gene in GBM that could play a driver role but without providing any functional information. Here, we have sequenced IRE1 exons in 23 human GBMs and identified a new somatic mutation on the IRE1 gene. Interestingly, using biochemical approaches and an orthotopic tumor graft mouse model we show that this variant displays an altered RIDD selectivity in particular towards the specific RIDD substrates already characterized in GBM (SPARC and PER1) and also miR-17, a previously described RIDD substrate whose expression has also a prognostic value in GBM. IRE1-dependent gene expression pattern alterations lead to major functional consequences in terms of tumor phenotype including (i) a magnified induction of the inflammatory and pro-angiogenic pathways based on transcriptome signatures and (ii) an increase of the aggressiveness/infiltration potential of U87 derived tumors in mice. This study provides the first evidence that RIDD substrates selectivity can be modulated and unravels the first mechanistic example of how a somatic mutation in the IRE gene can provide adaptive advantages to glioblastoma cells. Note: This abstract was not presented at the meeting. Citation Format: Eric Chevet. Selectivity of regulated IRE1 dependent decay of RNA controls pro-oncogenic signaling in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2015-1253

Ire1-mediated decay in mammalian cells relies on mRNA sequence, structure, and translational status.

Endoplasmic reticulum (ER) stress occurs when misfolded proteins overwhelm the capacity of the ER, resulting in activation of the unfolded protein response (UPR). Ire1, an ER transmembrane nuclease and conserved transducer of the UPR, cleaves the mRNA encoding the transcription factor Xbp1 at a dual stem-loop (SL) structure, leading to Xbp1 splicing and activation. Ire1 also cleaves other mRNAs localized to the ER membrane through regulated Ire1-dependent decay (RIDD). We find that during acute ER stress in mammalian cells, Xbp1-like SLs within the target mRNAs are necessary for RIDD. Furthermore, depletion of Perk, a UPR transducer that attenuates translation during ER stress, inhibits RIDD in a substrate-specific manner. Artificially blocking translation of the SL region of target mRNAs fully restores RIDD in cells depleted of Perk, suggesting that ribosomes disrupt SL formation and/or Ire1 binding. This coordination between Perk and Ire1 may serve to spatially and temporally regulate RIDD.

Cartilage-specific ablation of XBP1 signaling in mouse results in a chondrodysplasia characterized by reduced chondrocyte proliferation and delayed cartilage maturation and mineralization

To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage. Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation. Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR. Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease.

Endoplasmic reticulum stress response and transcriptional reprogramming.

Endoplasmic reticulum stress response and transcriptional reprogramming.

Ire1 Has Distinct Catalytic Mechanisms for XBP1/HAC1 Splicing and RIDD

SUMMARYAn evolutionarily conserved unfolded protein response (UPR) component, IRE1, cleaves XBP1/HAC1 introns in order to generate spliced mRNAs that are translated into potent transcription factors. IRE1 also cleaves endoplasmic-reticulum-associated RNAs leading to their decay, an activity termed regulated IRE1-dependent decay (RIDD); however, the mechanism by which IRE1 differentiates intron cleavage from RIDD is not well understood. Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase and mutations at oligomerization interfaces. Using an IRE1 RNase inhibitor, STF-083010, selective inhibition of XBP1 splicing indicates that XBP1 promotes cell survival, whereas RIDD leads to cell death, revealing modulation of IRE1 activities as a drug-development strategy.

The unfolded protein response in virus infections.

The unfolded protein response in virus infections.

Can't RIDD off viruses.

The mammalian genome has evolved to encode a battery of mechanisms, to mitigate a progression in the life cycle of an invasive viral pathogen. Although apparently disadvantaged by their dependence on the host biosynthetic processes, an immensely faster rate of evolution provides viruses with an edge in this conflict. In this review, I have discussed the potential anti-virus activity of inositol-requiring enzyme 1 (IRE1), a well characterized effector of the cellular homeostatic response to an overloading of the endoplasmic reticulum (ER) protein-folding capacity. IRE1, an ER-membrane-resident ribonuclease (RNase), upon activation catalyses regulated cleavage of select protein-coding and non-coding host RNAs, using an RNase domain which is homologous to that of the known anti-viral effector RNaseL. The latter operates as part of the Oligoadenylate synthetase OAS/RNaseL system of anti-viral defense mechanism. Protein-coding RNA substrates are differentially treated by the IRE1 RNase to either augment, through cytoplasmic splicing of an intron in the Xbp1 transcript, or suppress gene expression. This referred suppression of gene expression is mediated through degradative cleavage of a select cohort of cellular RNA transcripts, initiating the regulated IRE1-dependent decay (RIDD) pathway. The review first discusses the anti-viral mechanism of the OAS/RNaseL system and evasion tactics employed by different viruses. This is followed by a review of the RIDD pathway and its potential effect on the stability of viral RNAs. I conclude with a comparison of the enzymatic activity of the two RNases followed by deliberations on the physiological consequences of their activation.

Physiological roles of regulated Ire1 dependent decay.

Inositol-requiring enzyme 1 (Ire1) is an important transducer of the unfolded protein response (UPR) that is activated by the accumulation of misfolded proteins in the endoplamic reticulum (ER stress). Activated Ire1 mediates the splicing of an intron from the mRNA of Xbp1, causing a frame-shift during translation and introducing a new carboxyl domain in the Xbp1 protein, which only then becomes a fully functional transcription factor. Studies using cell culture systems demonstrated that Ire1 also promotes the degradation of mRNAs encoding mostly ER-targeted proteins, to reduce the load of incoming ER “client” proteins during ER stress. This process was called RIDD (regulated Ire1-dependent decay), but its physiological significance remained poorly characterized beyond cell culture systems. Here we review several recent studies that have highlighted the physiological roles of RIDD in specific biological paradigms, such as photoreceptor differentiation in Drosophila or mammalian liver and endocrine pancreas function. These studies demonstrate the importance of RIDD in tissues undergoing intense secretory function and highlight the physiologic role of RIDD during UPR activation in cells and organisms.

A Tale of Two Distinct Mechanisms for IRE1 RNase (752.9)

IRE1, an ER transmembrane receptor kinase, is a component of the Unfolded Protein Response (UPR) pathway, and acts as an endoribonuclease (RNase) to cleave the intron from XBP1/HAC1 mRNA coding an UPR‐specific transcription factor. Interestingly, recent reports have revealed that UPR also induces a decrease in levels of ER‐associated mRNAs, including INSULIN mRNA in an IRE1‐dependent manner1, which has been termed Regulated IRE1 Dependent Decay (RIDD). While XBP1/HAC1 splicing and RIDD are proposed to be carried out by IRE1, the mechanism by which IRE1 differentiates between XBP1/HAC1 and RIDD substrate mRNAs has not been investigated. Major differences in the cleavage of the HAC1/XBP1 intron and of RIDD RNA include RNA sequences at the cleavage sites, and that HAC1/XBP1 mRNA cleavage occurs at the ends of the intron whereas certain RIDD substrate RNA are cleaved at multiple sites. In addition, HAC1/XBP1 exon cleavage must be coordinated with tRNA ligase activity while RIDD RNA fragments should be shielded from tRNA ligase to promote their degradation.Upon development of a robust in vitro RIDD RNase assay with purified recombinant IRE1 expressed in baculovirus or E. coli, we examined the molecular mechanisms by which IRE1 cleaves either HAC1/XBP1 or RIDD RNA substrates. We have found that IRE1 engaged in cleavage of HAC1/XBP1 RNA cannot be competed by addition of cold RIDD RNA, while HAC1/XBP1 RNA can be effectively competed by excess cold HAC1/XBP1. The converse was true for IRE1 engaged in RIDD cleavage. Further analyses revealed that IRE1 oligomer constitutes the site for RNA substrate binding and catalysis, while IRE1 that acts on RIDD RNA does not require oligomerization. Our study provides a molecular basis for designing strategies to differentially activate (inhibit) one of the Ire1 RNase activities without affecting the other which may lead to new strategies for treating diseases associated with IRE1 activation.#1: Hollien, J., Science 313, 104 (2006), Han, D., Cell 138, 562 (2009)

Regulated IRE1‐dependent decay participates in curtailing immunoglobulin secretion from plasma cells

Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo-nuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory μ chains, which explains the reduction in secretory μ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.

Xbp1-Independent Ire1 Signaling Is Required for Photoreceptor Differentiation and Rhabdomere Morphogenesis in Drosophila

The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere andin the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the fatty acid transport protein (fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.

Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.