Pseudorabies Virus Infection Induces Endoplasmic Reticulum Stress Through PERK and IRE1 Pathways of Unfolded Protein Response

Pseudorabies virus (PRV) is a pathogen of swine resulting in devastating disease. Some viral infections can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) to restore ER homeostasis. However, the mechanism of how PRV induces ER stress and UPR activation remains unclear. Here, levels of proteins or transcriptional factors of three UPR pathways were examined in suspension-cultured BHK-21 cells to investigate PRV-induced ER stress. Results showed that PRV triggered ER stress and UPR of the host cells with the upregulated expression of glucose-related protein 78 kD and 94 kD (GRP78 and GRP94). The protein kinase RNA-like ER kinase (PERK) pathway was activated to upregulate ATF4, CHOP, and GADD34 expression. Additionally, the inositol requiring kinase 1 (IRE1) pathway was triggered by splicing of X box-binding protein 1 (XBP1) mRNA and the enhanced expression of p58IPK and EDEM1. Furthermore, our data demonstrated that PRV took advantage of ER stress to accelerate its replication with the activation of the PERK and IRE1 pathways in suspension-cultured BHK-21 cells, and the glycoprotein B played a crucial role in ER stress.

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a.

Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1a. While IRE1a can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1a complexes as well as the targeting mechanism that recruits XBP1 to IRE1a oligomers, remain unknown. Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1a-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1a clusters, which only assemble upon overexpression of fluorescently-tagged IRE1a during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1a assemblies that are homogenously distributed throughout the ER membrane.

YY1 and RTCB in mouse uterine decidualization and embryo implantation

As a multifunctional transcription factor, YY1 regulates the expression of many genes essential for early embryonic development. RTCB is an RNA ligase that plays a role in tRNA maturation and Xbp1 mRNA splicing. YY1 can bind in vitro to the response element in the proximal promoter of Rtcb and regulate Rtcb promoter activity. However, the in vivo regulation and whether these two genes are involved in the mother-fetal dialogue during early pregnancy remain unclear. In this study, we validated that YY1 bound in vivo to the proximal promoter of Rtcb in mouse uterus of early pregnancy. Moreover, via building a variety of animal models, our study suggested that both YY1 and RTCB might play a role in mouse uterus decidualization and embryo implantation during early pregnancy.

The epithelial-specific ER stress sensor IRE1β enables host-microbiota crosstalk to affect colon goblet cell development

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express IRE1β (Ern2), a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor IRE1α. How IRE1β functions at the host-environment interface and why a second IRE1 paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that IRE1β was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced IRE1β expression. IRE1β acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although IRE1α can also splice Xbp1 mRNA, it did not act redundantly to IRE1β in this context. By regulating assembly of the colon mucus layer, IRE1β further shaped the composition of the gut microbiota. Mice lacking IRE1β had a dysbiotic microbial community that failed to induce goblet cell development when transferred into germ-free wild type mice. These results show that IRE1β evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.

Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay

SUMMARYThe unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum(ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. We identified binding sites for the RNA-binding protein Pumilio in the 3’UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio bound both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced was stabilized by Pumilio. Furthermore, Pumilio displayed Ire1 kinase-dependent phosphorylation during ER stress, which was required for its stabilization of Xbp1spliced. Human IRE1 could directly phosphorylate Pumilio, and phosphorylated Pumilio protected Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an important and unexpected regulatory link between an RNA-binding protein and the UPR.

Involvement of miR-190b in Xbp1 mRNA Splicing upon Tocotrienol Treatment

We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA–miRNA–Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.

SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN), displaying symptoms overlapping with Shwachman-Diamond-Syndrome (SDS).1 Here, we investigate srp54 KO zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish are embryonically lethal and display, next to severe neutropenia, multi-systemic developmental defects. In contrast, srp54+/- zebrafish are viable, fertile and only show mild neutropenia. Interestingly, injection of human SRP54 mRNAs carrying mutations observed in patients (T115A, T117Δ and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the variable phenotypes observed in patients may be due to mutation-specific dominant negative effects on the functionality of the residual wildtype SRP54 protein. Consistently, overexpression of mutated SRP54 also induced neutropenia in wildtype fish and impaired granulocytic maturation of human promyelocytic HL-60 cells as well as of healthy cord-blood derived CD34+ HSPCs. Mechanistically, srp54 mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Vice-versa, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to SRP54 loss. The heterogenic phenotypes observed in patients, ranging from mild CN to SDS-like disease, may be due to different dominant negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.

Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1α Axis

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1α/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1α by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1α (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.

A Molecular Mechanism for Turning off IRE1α Signaling During Endoplasmic Reticulum Stress

SummaryMisfolded proteins in the endoplasmic reticulum (ER) activate IRE1α endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress and thereby attenuating IRE1α. Although sustained activation of IRE1α is linked to human diseases, it is not clear how IRE1α is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1α/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1α. This leads to inhibition of higher-order oligomerization and attenuation of IRE1α RNase activity during prolonged ER stress. In Sec63 deficient cells, IRE1α remains activated for a long time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to regulate the dynamics of IRE1α signaling in cells.

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs

Endoplasmic Reticulum (ER) stress is a hallmark of various diseases. Cells cope with ER stress through the activation of an adaptive signaling pathway named the Unfolded Protein Response (UPR) which is mediated by three ER-resident sensors. The most evolutionary conserved of the UPR sensors is IRE1α that elicits diverse downstream effects through its kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through Regulated IRE1-Dependent Decay (RIDD). The degree of exertion of these two activities plays an instrumental role in cells’ life and death decisions upon ER stress. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented, however, the precise mechanisms controlling whether IRE1 signaling is adaptive or pro-death (terminal) remain unclear. This prompted us to further investigate those mechanisms and we hypothesized that XBP1 mRNA splicing and RIDD activity could be co-regulated within the context of the IRE1α RNase regulatory network. We showed that a key nexus in this pathway is the tRNA ligase RtcB which, together with IRE1α, is responsible for XBP1 mRNA splicing. We demonstrated that RtcB is tyrosine phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we identified RtcB Y306 as a key residue which, when phosphorylated, perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing and favoring RIDD. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and depending on the nature of the stress determines cell adaptive or death outputs.