InteractiveFly: GeneBrief

Inositol-requiring enzyme-1: Biological Overview | References

Gene name - Inositol-requiring enzyme-1

Synonyms -

Cytological map position - 92B3-92B7

Function - enzyme, RNA splicing factor

Keywords - unfolded protein response, photoreceptor differentiation and rhabdomere morphogenesis, mediates the nonconventional splicing of an intron from X box binding protein 1

Symbol - Ire1

FlyBase ID: FBgn0261984

Genetic map position - chr3R:15679630-15687013

Classification - RNase domain (also known as the kinase extension nuclease domain) of Ire1, Protein kinase domain

Cellular location - ER transmembrane protein

NCBI link: EntrezGene
Ire1 orthologs: Biolitmine
Recent literature
Huang, H. W., Zeng, X., Rhim, T., Ron, D. and Ryoo, H. D. (2017). The requirement of IRE1-XBP1 in resolving physiological stress during Drosophila development. J Cell Sci. PubMed ID: 28775151
IRE1 mediates the Unfolded Protein Response (UPR) in part by regulating XBP1 mRNA splicing in response to endoplasmic reticulum (ER) stress. In cultured metazoan cells, IRE1 also exhibits XBP1-independent biochemical activities. IRE1 and XBP1 are developmentally essential genes in Drosophila and mammals, but the source of the physiological ER stress and the relative contributions of XBP1 activation versus other IRE1 functions to development remain unknown. This study employed Drosophila to address this question. Specifically, specific regions of the developing alimentary canal, fat body and the male reproductive organ are the sources of physiological stress that requires ire1 and xbp1 for resolution. In particular, the developmental lethality associated with xbp1 nulls was rescued by transgenic expression of xbp1 in the alimentary canal. IRE1's domains involved in detecting unfolded proteins, cleaving RNAs and activating XBP1 splicing were all essential for development. The earlier onset of developmental defects of ire1 mutant larvae compared to xbp1-null flies supports a developmental role for XBP1-independent IRE1 RNase activity while challenging the importance of RNase-independent effector mechanisms of Drosophila IRE1 function.
Mitra, S. and Ryoo, H. D. (2021). The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele. Dev Biol. PubMed ID: 34265355
Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. This study reports that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, it was found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1(H890A) homozygous mosaic eyes. Furthermore, the Ire1(H890A) mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. These results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.
Zhao, P., Huang, P., Xu, T., Xiang, X., Sun, Y., Liu, J., Yan, C., Wang, L., Gao, J., Cui, S., Wang, X., Zhan, L., Song, H., Liu, J., Song, W. and Liu, Y. (2021). Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila.. iScience 24(8): 102819. PubMed ID: 34381963
The endoplasmic reticulum (ER)-resident transmembrane protein kinase/RNase Ire1 is a conserved sensor of the cellular unfolded protein response and has been implicated in lipid homeostasis, including lipid synthesis and transport, across species. This study reports a novel catabolic role of Ire1 in regulating lipid mobilization in Drosophila. Ire1 is activated by nutrient deprivation, and, importantly, fat body-specific Ire1 deficiency leads to increased lipid mobilization and sensitizes flies to starvation, whereas fat body Ire1 overexpression results in the opposite phenotypes. Genetic interaction and biochemical analyses revealed that Ire1 regulates lipid mobilization by promoting Xbp1s-associated FoxO degradation and suppressing FoxO-dependent lipolytic programs. These results demonstrate that Ire1 is a catabolic sensor and acts through the Xbp1s-FoxO axis to hamper the lipolytic response during chronic food deprivation. These findings offer new insights into the conserved Ire1 regulation of lipid homeostasis.
Fangninou, F. F., Yu, Z., Li, Z., Guadie, A., Li, W., Xue, L. and Yin, D. (2023). Metastatic effects of environmental carcinogens mediated by MAPK and UPR pathways with an in vivo Drosophila Model. J Hazard Mater 441: 129826. PubMed ID: 36084456
Metastasis includes tumor invasion and migration and underlies over 90% of cancer mortality. The metastatic effects of environmental carcinogens raised serious health concerns. However, the underlying mechanisms remained poorly studied. In the present study, an in vivo Ras(V12)/lgl(-/-) model of the fruitfly, Drosophila melanogaster, with an 8-day exposure was employed to explore the metastatic effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126), perfluorooctanoic acid (PFOA) and cadmium chloride (CdCl(2)). At 1.0 mg/L, PCB126, PFOA, and CdCl(2) significantly increased tumor invasion rates by 1.32-, 1.33-, and 1.29-fold of the control, respectively. They also decreased the larval body weight and locomotion behavior. Moreover, they commonly disturbed the expression levels of target genes in MAPK and UPR pathways, and their metastatic effects were significantly abolished by the addition of p38 inhibitor (SB203580), JNK inhibitor (SP600125) and IRE1 inhibitor (KIRA6). Notably, the addition of the IRE inhibitor significantly influenced sna/E-cad pathway which is essential in both p38 and JNK regulations. The results demonstrated an essential role of sna/E-cad in connecting the effects of carcinogens on UPR and MAPK regulations and the resultant metastasis.
Yang, S., Jiang, H., Bian, W., Xu, W., Guo, Y., Song, S., Zheng, J., Kuang, X., Wu, C., Ding, X., Guo, X., Xue, L., Yu, Z., Zhang, Y., Ryoo, H. D., Li, X. and Ma, X. (2022). Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation. Proc Natl Acad Sci U S A 119(42): e2202133119. PubMed ID: 36215479
Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. This study reports that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans.
Zhao, N., Li, N. and Wang, T. (2023). PERK prevents rhodopsin degradation during retinitis pigmentosa by inhibiting IRE1-induced autophagy. J Cell Biol 222(5). PubMed ID: 37022709
Chronic endoplasmic reticulum (ER) stress is the underlying cause of many degenerative diseases, including autosomal dominant retinitis pigmentosa (adRP). In adRP, mutant rhodopsins accumulate and cause ER stress. This destabilizes wild-type rhodopsin and triggers photoreceptor cell degeneration. To reveal the mechanisms by which these mutant rhodopsins exert their dominant-negative effects, this study established an in vivo fluorescence reporter system to monitor mutant and wild-type rhodopsin in Drosophila. By performing a genome-wide genetic screen, PERK signaling was found to play a key role in maintaining rhodopsin homeostasis by attenuating IRE1 activities. Degradation of wild-type rhodopsin is mediated by selective autophagy of ER, which is induced by uncontrolled IRE1/XBP1 signaling and insufficient proteasome activities. Moreover, upregulation of PERK signaling prevents autophagy and suppresses retinal degeneration in the adRP model. These findings establish a pathological role for autophagy in this neurodegenerative condition and indicate that promoting PERK activity could be used to treat ER stress-related neuropathies, including adRP.

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. This study shows that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in 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. These 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 (Coelho, 2013).

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. When the folding capacity of the ER is impaired, the presence of incorrectly folded (misfolded) proteins in the ER causes ER stress and activates the unfolded protein response (UPR), which helps to restore homeostasis in the ER (Ron, 2007; Walter, 2011). In higher eukaryotes, the activation of the UPR is accomplished via three signaling pathways induced by ER-resident molecular ER stress sensors: protein kinase (PKR)-like ER kinase (PERK),activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (Ire1). Being conserved in all eukaryotes, Ire1 contains an ER luminal domain, which is involved in the recognition of misfolded proteins (Credle, 2005; Gardner, 2011), and cytoplasmic endoribonuclease and kinase domains, which are involved in the activation of downstream pathways. Activated Ire1 mediates the nonconventional splicing of an intron from X box binding protein 1 (Xbp1) mRNA (or HAC1 mRNA, the yeast Xbp1 ortholog), causing a frameshift during translation, thereby introducing a different carboxyl domain in the Xbp1 protein (Cox, 1996; Mori, 1996; Yoshida, 2001; Calfon, 2002; Shen, 2001). Xbp1spliced is an effective transcription factor that regulates the expression of ER chaperones and other target genes (Acosta-Alvear, 2007) (Coelho, 2013).

In addition to mediating Xbp1 mRNA splicing, cell culture studies demonstrated that Ire1 promotes the degradation of mRNAs encoding ER-targeted proteins, a process called RIDD (regulated Ire1-dependent decay), to reduce the load of ER client proteins during ER stress (Hollien, 2006; Han, 2009; Hollien, 2009). The cytosolic domain of mammalian IRE1 binds Traf2 (tumor necrosis factor receptor-associated factor 2; Urano, 2000), an upstream activator of the c-Jun N-terminal kinase (JNK) signaling pathway. This IRE1/ Traf2 interaction is also independent of Xbp1 splicing and may lead to the activation of apoptosis after prolonged ER stress (Yoneda, 2001; Coelho, 2013 and references therein).

In the Drosophila photoreceptor cells, the rhabdomere is the light-sensing organelle, a stack of photosensitive apical microvilli that is formed during the second half of pupal development. The rhabdomere is formed in the apical domain of each photoreceptor cell, which after a 90 rotation extends its apical domain along the proximal-distal axis of the retina. The growth of the rhabdomere requires the delivery of large amounts of membrane and proteins into this structure, imposing a considerable demand to the cellular mechanisms controlling protein folding and membrane production in the ER (Coelho, 2013).

Among the proteins targeted to the developing rhabdomeres are the rhodopsins, the light-sensitive proteins, and other proteins involved in the transduction of the light stimuli. Rhodopsin-1 (Rh1) is a seven transmembrane domain protein that starts to be expressed by 78% of pupal life and is delivered to the rhabdomeres of the outer photoreceptors (R1–R6), in a trafficking process that requires the activity of Rab11, MyosinV, and dRip11 (Li, 2007; Satoh, 2005). The delivery of Rh1 to the rhabdomere is required for rhabdomere morphogenesis because in Rh1-null mutants, the rhabdomere does not form, causing degeneration of the photoreceptors (Coelho, 2013).

In mammalians, the microRNA mir-708 is upregulated by CCAAT enhancer-binding protein homologous protein (CHOP) to control rhodopsin expression levels and prevent an excessive rhodopsin load into the ER (Behrman, 2011). In Drosophila, Ire1 signaling is activated in the photoreceptors upon expression of Rh1 folding mutants (Ryoo, 2007; Griciuc, 2010; Kang, 2009) or in ninaA mutations that cause the accumulation of misfolded Rh1 in the ER (Mendes, 2009). However, the role of Ire1 signaling during normal photoreceptor differentiation remains unknown. This study shows that Ire1 signaling is activated in the photoreceptors during pupal stages of Drosophila development. Ire1 mutant photoreceptors have defects in the delivery of Rh1 to the rhabdomere and the secretion of Spacemaker/Eyes Shut (Spam/Eys) into the interrhabdomeral space (IRS). Surprisingly, Xbp1-null mutant photoreceptors have a milder phenotype with no defects in Rh1 delivery into the rhabdomere or Spam/Eys secretion. Targets of RIDD are upregulated in Ire1 mutant retinas, including the fatty acid transport protein (fatp), a known regulator of Rh1 protein levels (Dourlen, 2012). Finally, it was shown that the regulation of fatp levels by RIDD is critical for normal Rh1 delivery into the rhabdomere (Coelho, 2013).

Studies in mammalian systems revealed that the Ire1/Xbp1 signaling pathway is important during development for the differentiation of secretory cells. For example, Xbp1 'knockout' mice have defects in the differentiation of antibody-secreting plasma cells (Reimold, 2001) and secretory cells of the exocrine glands of the pancreas (Lee, 2005). Presumably, in these cases, activation of Ire1/Xbp1 signaling is required to increase the capacity of the ER to fold and process the high load of secreted proteins (Coelho, 2013).

The present results demonstrate that Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis, a process that also imposes a high demand to the capacity of the ER to fold proteins such as Spam/Eys and Rh1. As shown, Ire1 mutant photoreceptors have defects in the secretion of Spam/Eys to the IRS and in the delivery of Rh1 to the rhabdomere. However, activation is seen of the Xbp1-EGFP reporter starting at 48 hr of pupal development, well before when the Spam/Eys secretion and Rh1 delivery defects are observed. Presumably, the folding of other unidentified proteins during these earlier stages might also require Ire1 signaling. It is noteworthy though, that in mutant B lymphocytes modified to lack antibody production, Ire1 is still activated (and Xbp1 spliced) upon lymphocyte differentiation to plasma cells. Activation of Ire1/Xbp1 signaling in this context seems to be part of the process of plasma cell differentiation, independently of the accumulation of misfolded proteins in the ER lumen (Coelho, 2013).

Ire1 function is also required for the regulation of the membrane lipids. In mammalians, Ire1/Xbp1 signaling regulates the biosynthesis of phospholipids and other lipids (Sriburi, 2007; Lee, 2008). A study in yeast demonstrated that Ire1 is activated by 'membrane aberrancy,' a condition of stress caused by the experimental depletion of inositol (Promlek, 2011). Activation of Ire1 in this case occurs by a mechanism that is distinct from the one involving the recognition of misfolded proteins by the luminal domain of Ire1 (Promlek, 2011). Furthermore, Ire1 can be activated by direct binding of flavonoids, such as quercetin, to a pocket present in the cytoplasmic domain of Ire1, in a mechanism that is also independent of the binding of misfolded proteins to Ire1 (Wiseman, 2010). The present results do not clarify if Ire1 activation in the photoreceptors during pupal stages results from the accumulation of misfolded proteins in the ER lumen or an imbalance in the membrane lipids (Coelho, 2013).

The results demonstrate that Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in an Xbp1-independent manner. Studies using cell culture paradigms demonstrated that, in addition to mediating Xbp1 mRNA splicing, Ire1 also promotes RIDD, the degradation of mRNAs encoding ER-targeted proteins (Hollien, 2006; Han, 2009; Hollien, 2009), but the physiological significance of the RIDD mechanism is unknown. Quantitative RT-PCR results show that RIDD targets are upregulated in Ire1 mutant eyes, including fatp, a regulator of Rh1 protein levels (Dourlen, 2012). The results show that regulation of fatp mRNA by RIDD is critical for rhabdomere morphogenesis because the experimental downregulation of fatp mRNA by RNAi rescues the Rh1 rhabdomere delivery defect observed in Ire1 mutants (Coelho, 2013).

Rh1 protein levels and Rh1 delivery to the rhabdomere are very sensitive to the levels of sphingolipids and phosphatidic acid. Increased fatp levels may lead to an increase in the levels of fatty acids and, subsequently, phosphatidic acid, which is known to downregulate Rh1 protein levels and cause rhabdomere morphogenesis defects (Raghu, 2009). High levels of phosphatidic acid disrupt the Arf1-dependent transport of membrane to the developing rhabdomere (Raghu, 2009). The results show that phosphatidic acid levels are elevated in Ire1 mutant retinas, and lowering phosphatidic acid levels by expression of LPP rescues the defects observed in Ire1 mutants, demonstrating that Ire1/fatp-dependent regulation of fatty and phosphatidic acids levels is important for rhabdomere morphogenesis in Drosophila. In addition, it is possible that the increase in phosphatidic acid levels in Ire1 mutant photoreceptors is also caused by the activation of PERK because upregulation was observed of the PERK pathway mediator ATF4 in Ire1 mutant photoreceptors, and in cell culture models, it was shown that PERK is able to phosphorylate diacylglycerol and generate phosphatidic acid (Bobrovnikova-Marjon, 2012). In conclusion, the results, using well-characterized genetic tools (Ire1 and Xbp1-null mutations) and a developmental paradigm (photoreceptor differentiation in the Drosophila pupa), demonstrate the physiological relevance of Xbp1-independent mechanisms downstream of Ire1 signaling (Coelho, 2013).

Ire1 supports normal ER differentiation in developing Drosophila photoreceptors

The endoplasmic reticulum (ER) serves virtually all aspects of cell physiology and, by pathways incompletely understood, is dynamically remodeled to meet changing cell needs. Inositol-requiring enzyme 1 (Ire1), a conserved core of the Unfolded Protein Response (UPR), participates in ER remodeling and is particularly required during the differentiation of cells devoted to intense secretory activity, "professional" secretory cells. This study characterize Ire1's role in ER differentiation in developing Drosophila compound eye photoreceptors (R cells). As part of normal development, R cells take a turn as professional secretory cells with a massive secretory effort that builds the photosensitive membrane organelle, the rhabdomere. Rough ER sheets proliferate as rhabdomere biogenesis culminates and Ire1 is required for normal ER differentiation. Ire1 is active early in R cell development and is required in anticipation of peak biosynthesis. Without Ire1, rough ER sheets are strongly reduced and the extensive cortical ER network at the rhabdomere base, the subrhabdomere cisterna (SRC), fails. Instead, ER proliferates in persistent, ribosome-poor tubular tangles. A phase of Ire1 activity early in R cell development thus shapes dynamic ER (Xu, 2016).

From its initial specification in the morphogenetic furrow, to its adult service as a photosensor, a developing Drosophila R cell plays many roles, calling upon a sequence of conserved, core cell functions that target the developmental task at hand. In the last quarter of pupal life that task is the enormous expansion of the photosensory membrane via secretory delivery of membrane rich in rhodopsin and allied elements of phototransduction. This study shows a corresponding expansion of stacked rER meets this secretory challenge; at this developmental stage, R cells are typical of cells devoted to intensive secretion, 'professional' secretory cells, generally. As seen with other professional secretory cells, this study found that R cells require Ire1 for normal ER expansion and differentiation. When normal cells proliferate rER stacks, Ire1 mutant ER proliferates in a dense, chaotic tangle of reticulon-rich tubules. The mechanisms by which Ire1 supports normal R cell ER differentiation remain unknown and in view of its multiple and far-reaching effectors are likely to be multiple. Shown in this study recapitulated in R cells, a common theme in Ire1 regulation of ER differentiation is a requirement in anticipation of secretory activity, suggesting Ire1 builds in secretory capacity as part of normal organelle programming. It is unlikely Ire1 acts alone and temporal overlap of Ire1 activity, shown in this study, and also by (Coelho, 2013), with that of PERK, a second conserved UPR pathway suggests they may cooperate in ER programming. The profuse tangle of reticulon-rich tubules that arises during peak secretory effort, resembles the pathology seen in Ire1 mutant yeast upon ER stress: in response to unfolded protein stress, normal yeast proliferate ER sheets but, although normal in the absence of stress, stressed Ire1 mutant yeast expand ER in dense, reticulon-rich tangles. Together, results suggest the possibility that Ire1 contributes essential shaping activity to a program of ER expansion and, in its absence, an unbalanced drive expands dysmorphic ER (Xu, 2016).

Shown in this study, and previously (Coelho, 2013), Ire1 loss compromises Rh1 production and secretory delivery needed to build the rhabdomere. Prior work has shown that Ire1's contribution to these tasks is Xbp1-independent, which is in accord with the current study, and now has been extended to ER shaping; R cell ER is normal in severe Xbp1 hypomorphs. Previously, it has been shown that Ire1 contributes to Rh1 delivery via degradation of fatty acid transporter, fatp, mRNA: Ire1 loss elevates fatp mRNA and its reduction using RNAi rescues Rh1 delivery (Coelho, 2013). As elevated levels of phosphatidic acid (PA) have been shown to disrupt R cell apical membrane transport, it is proposed that increased fatp elevates PA and thus degrades Rh1 delivery (Coelho, 2013). Abnormal, expanded rER is seen in R cells with increased PA, but appears distinct from the tangled tubular ER seen in this study. Tubulated ER seen in this study also differs from the dilated ER lumens commonly noted when misfolded secretory protein products over-accumulate, e.g., in Akita mice that accumulate misfolded proinsulin, and distinct from the strongly amplified, well-formed rER seen in ninaA mutants caused by lumenal Rh1 accumulation. Failure to assemble expanded rER sheets with accumulation of irregular, ribosome-poor ER tubules in Ire1 mutant R cells is reasonably the ultrastructural substrate for the severe reduction of Rh1 levels and growth deficit generally; without a definitive measure of reduced Rh1 synthesis, it is possible that reduced Rh1 levels are attributable to enhanced ER-Associated Degradation (ERAD) in an out of control ER. Loss of a normal SRC in Ire1 mutants plausibly contributes to the failure to deliver secretory traffic to the growing rhabdomere. Collapse of normal ER morphology is thus catastrophic for multiple cell activities (Xu, 2016).

The mechanism by which lower temperature rescues ER differentiation in Ire1 mutant cells remains to be determined, but may be connected to numerous observations showing basal, Ire1-independent, ER capacity supports many aspects of normal cell development and physiology. Indeed, despite profound ER disruption during peak rhabdomere growth, mutant R cells are viable and execute a wide range of normal cell physiologies, including normal cell polarity, cell fate specification and the complex choreography that assembles ommatidia. Similarly, Ire1 null nematodes are viable and morphologically normal, but die when challenged with mutations in other UPR branches or tunicamycin. Mouse hepatocytes lacking Ire1 α show reduced rER content but are otherwise phenotypically normal; however upon ER stress they fail to maintain lipid homeostasis. The gut of mice lacking Ire1beta is phenotypically normal, but sensitized to experimental colitis. In the absence of stress, ER morphology is normal in yeast ire1 mutants. It is speculated that when pupal development is slowed more than, twofold, 9-10 days at 19°C, versus 4 days at 25°C, basal ER capacity meets secretory demand (Xu, 2016).

The distribution of peripheral ER between sheet and tubule domains has been likened to a "tug-of-war", with activity promoting ER sheets poised against tubule promoting activity, particularly that of membrane curvature-inducing reticulons. The near disappearance of rER sheets and concomitant emergence of dense tubular tangles in Ire1 mutants suggests a runaway win for tubule promotion, potentially due to a failure of sheet-forming activities, an abnormal regulation of tubule formation, or a combination of both. Ribosomes promote ER sheets and it is possible the loss ribosomes in mutant R cells destabilizes rER. Alternately, abnormal, dense Rtnl1 accumulations seen in mutant R cells may reflect a cannibalization of ER sheets by misregulated Rtnl1 with a consequent reduction in Rh1 synthesis; misregulated Rtnl1 could also account for the loss of the SRC cortical ER network. The dynamic, stereotyped differentiation of ER morphology underlying Drosophila R cell differentiation presents a genetically accessible system to investigate how developmental programs shape ER (Xu, 2016).

Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. This study addresses which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt (Zhang, 2021).

Cell compartmentalization is not only mediated by membrane-bound organelles. It also relies on non-membrane bound biomolecular condensates (so-called membraneless organelles) that populate the nucleus and the cytoplasm (Zhang, 2021).

The formation of membraneless organelles has been shown to occur through phase separation, which can be driven by stress (such as ER, oxidative, proteostatic or nutrient stress), resulting in the formation of stress assemblies. Those are mesoscale coalescence of specific and defined components that phase separate. For instance, nutrient stress leads to the formation of many biocondensates. Most of them are RNA based, such as stress granules and P-bodies, but some are not. This is the case for glucose-starved yeast where metabolic enzymes foci and proteasome storage granules form, as well as Drosophila S2 cells that form Sec bodies under conditions of amino acid starvation (Zhang, 2021).

Sec bodies are related to the inhibition of protein secretion in the early secretory pathway. The early secretory pathway comprises the endoplasmic reticulum (ER), where newly synthesized proteins destined to the plasma membrane and the extracellular medium are synthesized. Proteins exit the ER at the ER exit sites (ERES) to reach the Golgi. The ERES are characterized by the concentration of COPI-coated vesicles whose formation requires six proteins, including Sec12 and Sar1, the inner coat proteins Sec23 and Sec24, and the outer coat proteins Sec13 and Sec31 (Gomez-Navarro, 2016). In addition, a larger hydrophilic protein called Sec16, has been identified as a key regulator of the ERES organization and COPII vesicle budding. Many additional lines of evidence support the role of Sec16 in optimizing COPII-coated vesicle formation and export from the ER (Zhang, 2021).

Upon the stress of amino acid starvation in Krebs Ringer bicarbonate buffer (KRB), the ERES of Drosophila S2 cells are remodeled into large round non-membrane bound phase-separated Sec bodies. They are typically observed by immunofluorescence after staining of endogenous Sec16, Sec23 and expressed Sec24-GFP. Importantly, Sec bodies are very quickly resolved upon stress relief (addition of growth medium). Finally, they appear to protect the components of the ERES from degradation and they help cells to survive under conditions of amino acid shortage (Zhang, 2021).

Phase separation has been shown to be driven by specific components, the so-called drivers, either RNAs or proteins harboring structural features that become exposed or modified under certain conditions. In the case of Sec bodies, Sec24AB and Sec16 have been shown to drive Sec body coalescence in a manner that depends on a small stretch of 44 residues in Sec16 and on the mono-ADP-ribosylation enzyme by PARP16. This illustrates the critical role of post-translational modifications in phase separation (Zhang, 2021).

In parallel, changes in cytoplasmic biophysical properties have also been shown to be important in phase separation, such as a drop of cytoplasmic pH within minutes, without post-translational modifications (Zhang, 2021).

This study sought out to (1) identify the pathways elicited in S2 cells upon incubation in the starvation medium KRB that lead to Sec body formation, and (2) to assess whether changes in the cytoplasmic biophysical properties play a role in the phase transition leading to Sec body formation. Amino acid starvation in KRB is shown to stimulate ER stress and activation of two downstream kinases, IRE1 and PERK (also known as PEK in flies) leading to the stimulation of the unfolded protein response (UPR). However, the sole activation of the IRE1 and PERK does not lead to Sec body formation. To form Sec bodies in KRB, IRE1 and PERK activation needs to be combined with a moderate salt stress. Accordingly, KRB incubation is faithfully mimicked by cell incubation with dithiothreitol (DTT) and addition of 100 mM NaCl. Interestingly, a high-salt stress addition of 150 mM NaCl, which activates the salt-inducible kinases (SIKs; SIK2 and SIK3), is sufficient to efficiently drive Sec body formation. Importantly, it was found that a decrease in the cytoplasmic ATP concentration, a general RNA degradation and the stimulation of the UPR are factors strongly correlated to Sec body formation (Zhang, 2021).

This study shows that the Sec bodies that form in Drosophila S2 cells incubated in KRB are fully recapitulated by activation of SIKs, IRE1 and PERK (through SCH100 plus DTT), leading to the activation of a downstream UPR. Strikingly, the strong activation of SIKs in (SCH150) also induces the UPR and leads to Sec body formation. The resulting structures in each condition appear to be similar in size and number, and their formation is reversible. Whether their content is strictly similar has not been addressed in this study (Zhang, 2021).

Taken together, the results show that Sec body formation requires the stimulation of two main signaling pathways. The first is the salt stress pathway (addition of 150 mM NaCl), which activates the SIKs in a necessary and sufficient manner. It also does not lead to a change in the cytoplasmic ATP concentration. It does induce RNA degradation and it stimulates the UPR in an unexpected manner, given that PERK and IRE1 inhibitors do not alter SCH150 driven Sec body formation (Zhang, 2021).

The second pathway is the activation of IRE1 and PERK (but not ATF6), downstream of ER stress, which is partly induced by the absence of amino acids in KRB. Activation of either IRE1 or PERK is necessary but not sufficient. To form Sec bodies, this activation needs to be combined with a moderate salt stress. It is proposed that IRE1 and PERK activation combined with SIK activation occur in KRB, which is recapitulated by SCH100 plus DTT. This is associated with a decrease in the cytoplasmic concentration of ATP, with RNA degradation and with a stimulation of the UPR (Zhang, 2021).

Interestingly, both strong salt stress (SCH150) and KRB lead to the activation of the UPR (measured by the increase in Bip protein level), leading to the possibility that SIKs, IRE1 and PERK interact with and/or activate, each other. Either IRE1 and/or PERK activate the SIKs, or SIK activation activates IRE1 and/or PERK. This still needs to be refined. The prominent role of salt stress and SIKs in remodeling the cytoplasm Strong salt stress is induced by a 4-fold increase of Na+ in the medium combined with bicarbonate. This triggers an increase of Na+ in the cytoplasm that activates one or more SIK (as shown by the phosphorylation of the SIK target HDAC4). Accordingly, SIK inhibition decreases Sec body formation (Zhang, 2021).

Keeping intracellular Na+ as near as possible to physiological concentrations (5 mM) is critical for cellular life, and the cell spends 40% of its available ATP to extrude Na+ against K+ with the NaK ATPase. It is therefore not surprising that Na+ stress would elicit a cytoprotective response, such as prominent as Sec body formation (and stress granule formation in mammalian cells). This will need to be further elucidated, as many organisms and tissues are subjected to increased circulating Na+. This study shows, however, that it is not equivalent to an osmotic shock and that this addition of salt does not lead to a decrease in a cell volume. In contrast to P-bodies in yeast, osmotic stress does not induce Sec bodies. Interestingly, Na+/salt stress has recently been shown to induce the biogenesis of the lysosomal pathway (i.e. more endo/lysosomes as well as an increase in its activity) via TFEB and TOR (Zhang, 2021).

Increased Na+ activates the intracellular Na+-sensor network revolving around the SIKs. The SIKs belong to the family of AMPKs, and have been shown to be part of a nutrient-sensing mechanism so far revolving around glucose and unbalance of the ATP-to-ADP ratio. In mammals, there are three genes encoding SIK (SIK1-SIK3) but only 2 in Drosophila. Drosophila SIK2 is the ortholog of human SIK1 and SIK2, and has been shown to have a link to the fly Hippo pathway, possibly linking nutrient to growth. Drosophila SIK3 is required for glucose sensing in the fly. Which SIK is involved in Sec body formation has not been clarified, as overexpression of each SIK individually has not proven enough to trigger Sec body formation, even when combined to some excess salt (SCH84 or SCH100). However, at least two SIKs appear to change their intracellular localization in KRB, that is, SIK2 and the long SIK3 isoform, which appear to cluster near the plasma membrane and localize to the nuclear envelope. The role of SIKs in the formation of stress assemblies (here the Sec bodies) appears important and novel, and needs to be investigated further. Other members of the AMPK family do not appear to be involved and changing the ratio ADP-to-ATP did not alter Sec body formation in the SIC system (Zhang, 2021).

Although a high salt stress is sufficient to trigger Sec body formation, the Sec body formation observed during incubation in the amino acid starvation buffer KRB elicits another pathway, the ER stress pathway, leading to the activation of both IRE1 and PERK. Indeed, KRB-induced Sec body formation is entirely mimicked by a moderate salt stress (SCH100) combined with activation of IRE1 and PERK induced by DTT (SCH100 plus DTT) (Zhang, 2021).

Surprisingly, this study found that salt stress as well as KRB induces the UPR, which this study found is a common downstream event in all conditions inducing Sec body formation. How salt stress activates the UPR and the exact role of IRE1 and PERK is still not fully understood. It does not appear to be via SIKs, as HG does not modulate the UPR, yet strongly reduces Sec body formation. Conversely, IRE1 and PERK inhibitors do not influence SCH150-induced Sec body formation, so the exact link between IRE1, PERK, SIKs and the UPR remains to be further investigated (Zhang, 2021).

How UPR stimulation induces Sec body formation is not completely understood. It could occur through the clustering of IRE1 and PERK, two membrane kinases, and them forming a template in the plane of the ER membrane. In this regard, UPR stimulation has been linked to the MARylation enzyme Drosophila PARP16, which is also a transmembrane protein of the ER that undergoes a remodeling in KRB. What other roles are played by events downstream of the UPR, for example, Bip upregulation itself, and cytoplasmic changes, remains to be elucidated in detail. One interesting aspect is whether the activation of UPR might affect biophysical properties, membrane association dynamics, and conformation and post-translational modifications of Sec16 and COPII subunits, which would lead to their enhanced phase separation properties under stress (Zhang, 2021).

Lowering the cytoplasmic ATP concentration is one parameter that induces Sec body formation The lowering of the cytoplasmic ATP concentration occurs in KRB and SCH100 plus DTT. Forcing this lowering or preventing it has a strong incidence on Sec body formation. This finding is consistent with the hydrotropic property of ATP, that is, it being able to prevent phase separation in the cytoplasm. At least in vitro, addition of 8 mM ATP dissolves or prevents the phase separation of purified FUS and TAF15. This is a concentration matching physiological range of ATP level in mammalian cells (1-10 mM) and that is also compatible with the S2 cell ATP concentration (1.7 mM) (Zhang, 2021).

One possibility to explain the decrease of ATP concentration is the activation of kinases (among them, IRE1, PERK and SIK) that would consume the ATP pool. However, it is proposed that the decrease in the intracellular ATP concentration is upstream of the kinase activation. Indeed, incubating cells in KRB induces a ROS shock (as this study showed experimentally), which could damage mitochondrial respiration, resulting, in turn, in ER stress, and UPR activation. Taken together, although this study unraveled a strong causality between low ATP and Sec body formation, the noticeable exception of such formation in SCH150 suggests other possibilities (Zhang, 2021).

In conclusion, this work illustrates the complexity of amino acid starvation, the number of pathways it activates and how they interact with each other, as well as the different cellular cytoplasmic biophysical parameters it affects. It is proposed that the formation of Sec bodies depends on the activation of signaling pathways leading to activation of SIKs, IRE1 and PERK, altogether leading to the activation of the UPR, one of the common features of all pathways leading to Sec body formation (Zhang, 2021).

Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila xbp1

The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER) stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions [the hydrophobic region 2 (HR2) and the C-terminal translational pausing site] present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. This study generated a Drosophila Xbp1 deletion mutant (Excision101) lacking both HR2 and C-terminal region, but not the Ire1 splicing site. Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. The results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced (Coelho, 2014).

A modified UPR stress sensing system reveals a novel tissue distribution of IRE1/XBP1 activity during normal Drosophila development

Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, previously reported XBP1 stress sensing systems (Souid, 2007; Ryoo, 2007), were modified based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA (Aragon, 2009; Yanagitani, 2009; Yanagitani, 2013). The improved XBP1 stress sensing system allowed detection of new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide novel insights into the brain homeostasis regulated by the UPR (Sone, 2013).

Inadequate sensitivity of existing XBP1 stress sensing systems can be overcome by improving the efficiency of unconventional splicing of xbp1 mRNA. Recent reports regarding the cellular localization of XBP1/HAC1 mRNA during its splicing allowed construction of a highly sensitive HG stress indicator that can visualize the activation of IRE1/XBP1 pathway at the third instar larval stage during normal Drosophila development. Several types of cells in the organs where IRE1/XBP1 activity was detected are known for having high secretory capacity (Sone, 2013).

In the larval brain, significant IRE1/XBP1 activity was found in glial cells. While glia had been originally thought to function as the structural support cells in the nervous system, it has been revealed that they play several important roles in the development and homeostasis of the nervous system. In Drosophila, glial cells are classified into three classes (surface-, cortex-, and neuropil-associated glia), each of which is subdivided further morphologically. Whether IRE1/XBP1 active glia is restricted to only a subtype of those glia, or more broadly, is currently under investigation (Sone, 2013).

In mammals, oligodendrocytes in the central nerve system and Schwann cells in the peripheral nerve system myelinate axons by producing a large amount of myelin membrane proteins, cholesterol, and membrane lipids through the secretory pathway. Recent reports suggested that ER stress in myelinating cells is important in the pathogenesis of various disorders of myelin. Neuropil glia and peripheral glia in Drosophila are the counterparts of oligodendrocytes and Schwann cells, respectively. Therefore, these cells are the candidates that show constitutive IRE1/XBP1 activity. Although Drosophila glia do not generate myelin sheaths, they form multi-layered membrane sheaths around neurons that are morphologically similar to the myelin sheaths in mammals. Thus, it is possible that the IRE1/XBP1 active glia protect neurons from their deterioration through this ensheathment, thereby contributing to brain homeostasis. Further studies are expected to be informative as to the pathological significance of IRE1/XBP1 functions in human glia (Sone, 2013).

IRE1/XBP1 pathway does not appear to be active in neuron. However, the possibility of neuronal IRE1/XBP1 activation in the brain was not excluded. In fact, slight neuronal IRE1/XBP1 activity was occasionally observed in the ventral nerve cord during repeated experiments. In this study, it is concluded that in the third instar larval brain, the IRE1/XBP1 pathway is predominantly activated in glia while the activation is not detectable in neurons (Sone, 2013).

The importance of IRE1/XBP1 activity in the gut has already been studied in Caenorhabditis elegans and mammals. Intra-tissue distribution of IRE1/XBP1 activity was detected in the proventriculus region of the gut. In the larval midgut and hindgut, an irregular distribution of IRE1/XBP1 active cells was observed. These were not entero-endocrine cells, as they did not colocalize with anti-Prospero antibody that marks those cells. Secretory intestinal cells in the midgut other than entero-endocrine cells including the intestinal stem cells are possible candidates for these IRE1/XBP1 active cells (Sone, 2013).

IRE1/XBP1 activity in the fly Malpighian tubules (analogous to the kidney in mammals) was also unexpected. The activity was detected throughout the organ, but not all of the cells were IRE1/XBP1 active. Although the Malpighian tubules are attached at the junction of the midgut and the hindgut, they are morphologically and functionally independent from both of them. Identification of the IRE1/XBP1 active cells in the gut and the Malpighian tubules might reflect a shared physiological function of both organs. One possible shared function may be the selective uptake of the essential molecules, including several metal ions, from the contents passing through those organs. IRE1/XBP1 pathway might regulate the function of some transporter channels in these organs. Drosophila Malpighian tubules are expected to be one of the models for the mammalian diabetic kidney diseases that are associated with UPR activation (Sone, 2013).

In this study, IRE1/XBP1 activity was also detected in the trachea. Previous reports suggest its relevance to glial IRE1/XBP1 activity. One of them showed that tracheal development in Drosophila brain was controlled by signals from glia. According to the report, the branches of cerebral trachea grow around the neuropile. If IRE1/XBP1 active glia were neuropile-associated glia, assessing IRE1/XBP1 activity at neuropile-associated glia is likely reveal the shared physiological function of IRE1/XBP1 pathway between brain and trachea. The other report, using embryonic trachea, indicated that the proper combination of secretory activity and endocytotic activity was importaAnt for the maturation of trachea as an airway. In tracheal maturation, Sar1, one of the core COPII proteins, was required for the secretion of protein, the luminal matrix assembly, and the following expansion of tube diameter to avoid the clogging of protein, while Rab5, the small GTPase that regulates the early stage of endocytosis, was required for the clearance of deposited materials in the lumen. It can be predicted that, even in larval trachea, IRE1/XBP1 pathway plays a crucial role in tracheal maturation by supplying the properly folded proteins to the transport machinery. In that case, in view of second instar larval lethality of xbp1-/- hypomorph mutant, it could also be hypothesized that the tracheal maturation/maintenance is still important for larval lethality, in addition to its importance for the embryonic development (Sone, 2013).

IRE1/XBP1 activity in the salivary gland has already been reported in a previous study (Souid, 2007). The salivary gland is commonly used for the determination of the subcellular localization of the protein in Drosophila cells due to its morphological features. The nuclear localization of HG indicator, XBP1-EGFP molecule, was clearly indicated. In addition, weak IRE1/XBP1 activity was detected in the fat body; it was attached to the salivary gland. Generally, the Drosophila fat body, which is equivalent to mammalian adipose tissue, functions as the organ for energy/lipid storage and is distributed throughout the larval body (Sone, 2013).

In addition to the larval tissues, IRE1/XBP1 activity was analyzed in the adult male reproductive organs. Though the previous RT-PCR study by Souid (2007) suggested the activity in the testis, the areas were IRE1/XBP1 activity was detected were the accessory glands and a limited area of the testis close to the testicular duct. In the accessory gland, seminal fluid containing several hormones, which facilitate reproductive traits such as sperm transfer, sperm storage, female receptivity, ovulation, and oogenesis, are produced and secreted. There are two morphologically distinct secretory cell types in Drosophila accessory gland. Ninety-six percent of the secretory cells are categorized as main cells and the others are secondary cells. Based on the intra-tissue distribution of IRE1/XBP1 active cells in the accessory gland, the active cells are likely to be main cells. Since each of these cell types expresses a unique set of genes, the confirmation of IRE1/XBP1 active cell type is expected to allow ingnarrow down the proteins related to IRE1/XBP1 activity. IRE1/XBP1 pathway is likely to function, to some extent, in maintaining proper fertility (Sone, 2013).

On the other hand, a possibility is considered that the EGFP signal detected in each organ might not necessarily reflect the unconventional splicing of xbp1-EGFP mRNA. Higher concentrations of the spliced xbp1-EGFP mRNA and resulting XBP1-EGFP in the cells induced by the Gal4/UAS system might cause the artifactual EGFP signal. The possibility was excluded that the EGFP signal in this study was detected independently of the unconventional splicing, based on the results in this study and the following reasoning (Sone, 2013).

There are two possible molecular mechanisms that cause the artifactual EGFP signal which is not derived from the unconventional splicing of xbp1-EGFP mRNA. One is the generation of EGFP or abnormal EGFP fusion proteins, resulting from translation initiation at the start codon of the EGFP coding sequence or at ATG codons coding Met residues in XBP1(s), respectively. The other is the proteolytic digestion of XBP1-EGFP fusion protein at the junction of XBP1 and EGFP portions. Both of these are prone to happen upon overexpression of fusion proteins in cells. In particular, the proteolytic digestion is often observed in the overexpression of GST fusion protein in Escherichia coli (Sone, 2013).

In this system, there is no nuclear localization signal (NLS) on the EGFP molecule. In contrast, xbp1 gene carries a NLS coding sequence located upstream of the unconventional splice site. There are a total of 11 ATG codons that code the Met residues of XBP1(s) molecule. Eight of the ATG codons are located downstream of NLS coding sequence. Therefore, due to the lack of NLS, both EGFP and the EGFP fusion proteins using these eight ATG codons as start codons should diffuse all over the cell upon their synthesis, if they were generated. EGFP signal was detected exclusively in the nucleus in the salivary gland, which is often used for the analysis of the cellular localization of the proteins in Drosophila. Hence, it is not reasonable to conclude that either EGFP or the possible eight EGFP fusion proteins above were expressed in the cells. Only the EGFP fusion proteins that use the other three ATG codons that are upstream of the NLS as start codons should be synthesized upon unconventional splicing and localized in the nucleus. The estimated molecular weights of those fusion proteins are 73.7, 74.3, and 80.0 kDa, respectively. Only a significant single band that represented the intact XBP1-EGFP (80.0 kDa) was detected in the S2 cell extract, in which XBP1-EGFP was overexpressed through the Gal4/UAS system. Therefore, it is concluded that the EGFP fusion protein synthesized in this study was the intact XBP1-EGFP (Sone, 2013).

Additionally, there are several ATG codons that are located upstream of the unconventional splice site and are in frame with EGFP coding sequence on unspliced xbp1 mRNA. However, inside the 23 bp of unconventional-spliced fragment, there is a TGA stop codon that is also in frame with EGFP coding sequence. Even if the translation initiated from these start codons, the synthesis of these products should be terminated at this TGA stop codon before the ribosome would reach the EGFP coding region (Sone, 2013).

Regarding the proteolytic digestion of XBP1-EGFP fusion protein, the resulting EGFP should diffuse all over the cell upon its synthesis due to the lack of NLS. Therefore, the possibility of the proteolytic digestion is also excluded based on the same reasoning as above. Taken together, it is concluded that the detected EGFP signal in this study exclusively reflected the occurrence of unconventional splicing of xbp1-EGFP mRNA (Sone, 2013).

In summary, this study improved the sensitivity of the XBP1 stress sensing system and newly identified several organs where IRE1/XBP1 pathway is constitutively activated under normal physiological conditions. In particular, in the larval brain, significant glial specific activation was detected. The improved system is expected to provide with a number of clues to reveal the molecular mechanisms underlying the normal development and homeostasis controlled by IRE1/XBP1 pathway (Sone, 2013).

Ire1 regulated XBP1 mRNA splicing is essential for the unfolded protein response (UPR) in Drosophila melanogaster

The ability of cells to survive and recover from deteriorating effects of endoplasmic reticulum (ER) stress relies on the unfolded protein response (UPR). The signaling pathway of Ire1p mediate mRNA splicing plays a divergant role in UPR responses in different organisms from yeast to mammals. This report that Ire1p mediated XBP1 mRNA splicing mechanism is extremely conserved and exerts a critical role for modulating Xbp1 protein synthesis in Drosophila melanogaster. This system is operative in Drosophila S2 cells as a prominent mechanism to mediate transcriptional activation of UPR responsive genes during ER stress (Plongthongkum, 2007).

Transcriptional activation of genes encoding molecular chaperones and folding enzymes during ER stress is a major response in eukaryotic cells to mount the stress for their survival. This study characterized the UPR pathway in Drosophila S2 cells. Drosophila Xbp1 is a bzip transcription factor that showed a very low homology in lysine, leucine rich region to its counterparts in mammal or worm. Interestingly, its coding sequence is organized in a highly conserved fashion. The fly XBP1 mRNA contains two overlapping ORFs. The ORF2 is in +1 frame relative to the ORF1. These ORFs were joined into the same reading frame during ER stress prior translation by eliminating the 23 nucleotide intron through an unusual splicing mechanism. The nucleotide sequence surrounding this intron showed extremely conserved feature of two 7 nucleotides ring stem loop structures critical for Ire1 mediated splicing in mammalian cells. This reveals that the stress regulated mRNA splicing pathway by Ire1p is remarkably conserved through evolution (Plongthongkum, 2007).

The finding that the endogenous XBP1u mRNA in S2 cells was not translated into protein but it was efficiently translated in transiently transfected cells providing an intriguing observation. The ORF2 located further down stream of the 5' end of the mRNA was simultaneously translated into protein implicating that the presence of the intron may form secondary structure which facilitates internal ribosome entry resembling Internal Ribosome Entry Site (IRES) of several RNA viruses. Currently there is no explanation why this structure is not functional in the Drosophila cells. It is possible that S2 cells produce specific factor(s) which bind and suppress the translation of the XBP1u mRNA. Albeit translated, the protein from ORF1 or ORF2 alone exhibits no activity. It is most likely that the two regions serve different functions such as DNA binding domain and transcription activation domain that must be linked to function (Plongthongkum, 2007).

While the mammalian UPR is a complex coordinating multiple mechanisms of Ire1/XBP1, ATF6, and PERK, the UPR in Drosophila S2 cells appears simpler, by relying mainly on Ire1/XBP1 pathway. Suppression of either IRE1 or XBP1 by RNAi completely abolished the UPR response. Nonetheless, this pathway might not be a simple linear signaling cascade. Besides inducing genes enriched ER-related processes during the stress, recent study has demonstrated additional functions of the insect UPR in mediating rapid repression of ER-targeted mRNAs. Such functions requires only Ire1 activity but are independent of Xbp1. Hence this system would be an interesting model for further elucidating the mechanism detail and physiological significant of metazoan Ire1/XBP1 cascade (Plongthongkum, 2007).

Functions of Ire1 orthologs in other species

Mutations in nonessential eIF3k and eIF3l genes confer lifespan extension and enhanced resistance to ER stress in Caenorhabditis elegans

The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. This study shows that loss-of-function mutations in the genes encoding the conserved eIF3k (see Drosophila CG10306) and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16 (see Drosophila foxo). Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1 (see Drosophila Ire1), ATF-6 (see Drosophila Atf6), and PEK-1 (see Drosophila PEK), and independent of DAF-16. These data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging (Cattie 2016).

Reduced Insulin/Insulin-like growth factor receptor signaling mitigates defective dendrite morphogenesis in mutants of the ER stress sensor IRE-1

Using the multidendritic arbor of PVD somatosensory neurons in the nematode Caenorhabditis elegans, this study identified a mutation in the ER stress sensor IRE-1/Ire1 (inositol requiring enzyme 1) (see Drosophila Ire1) as crucial for proper PVD dendrite arborization in vivo (see Drosophila axonogenesis). Regulation of dendrite growth in cultured rat hippocampal neurons depends on Ire1 function, showing an evolutionarily conserved role for IRE-1/Ire1 in dendrite patterning. PVD neurons of nematodes lacking ire-1 display reduced arbor complexity, whereas mutations in genes encoding other ER stress sensors display normal PVD dendrites, specifying IRE-1 as a selective ER stress sensor (see Drosophila as a model for ER stress and Drosophila UPR) that is essential for PVD dendrite morphogenesis. Although structure function analyses indicates that IRE-1's nuclease activity is necessary for its role in dendrite morphogenesis, mutations in xbp-1 (see Drosophila Xbp1), the best-known target of non-canonical splicing by IRE-1/Ire1, do not exhibit PVD phenotypes. Secretion and distal localization to dendrites of the DMA-1/leucine rich transmembrane receptor (DMA-1/LRR-TM) (see Drosophila rdo) is defective in ire-1 but not xbp-1 mutants, suggesting a block in the secretory pathway. Interestingly, reducing Insulin/IGF1 signaling (see Drosophila insulin signaling) can bypass the secretory block and restore normal targeting of DMA-1, and consequently normal PVD arborization even in the complete absence of functional IRE-1. This bypass of ire-1 requires the DAF-16/FOXO (see Drosophila foxo) transcription factor. In sum, this work identifies a conserved role for ire-1 in neuronal branching, which is independent of xbp-1, and suggests that arborization defects associated with neuronal pathologies may be overcome by reducing Insulin/IGF signaling and improving ER homeostasis and function (Salzberg, 2017).


Search PubMed for articles about Drosophila Ire1

Acosta-Alvear, D., Zhou, Y., Blais, A., Tsikitis, M., Lents, N. H., Arias, C., Lennon, C. J., Kluger, Y. and Dynlacht, B. D. (2007). XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Mol Cell 27: 53-66. PubMed ID: 17612490

Aragon, T., van Anken, E., Pincus, D., Serafimova, I. M., Korennykh, A. V., Rubio, C. A. and Walter, P. (2009). Messenger RNA targeting to endoplasmic reticulum stress signalling sites. Nature 457: 736-740. PubMed ID: 19079237

Behrman, S., Acosta-Alvear, D. and Walter, P. (2011). A CHOP-regulated microRNA controls rhodopsin expression. J Cell Biol 192: 919-927. PubMed ID: 21402790

Bobrovnikova-Marjon, E., Pytel, D., Riese, M. J., Vaites, L. P., Singh, N., Koretzky, G. A., Witze, E. S. and Diehl, J. A. (2012). PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation. Mol Cell Biol 32: 2268-2278. PubMed ID: 22493067

Calfon, M., Zeng, H., Urano, F., Till, J. H., Hubbard, S. R., Harding, H. P., Clark, S. G. and Ron, D. (2002). IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415: 92-96. PubMed ID: 11780124

Cattie, D.J., Richardson, C.E., Reddy, K.C., Ness-Cohn, E.M., Droste, R., Thompson, M.K., Gilbert, W.V. and Kim, D.H. (2016). Mutations in nonessential eIF3k and eIF3l genes confer lifespan extension and enhanced resistance to ER stress in Caenorhabditis elegans. PLoS Genet 12: e1006326. PubMed ID: 27690135

Coelho, D. S., Cairrao, F., Zeng, X., Pires, E., Coelho, A. V., Ron, D., Ryoo, H. D. and Domingos, P. M. (2013). Xbp1-independent ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila. Cell Rep 5: 791-801. PubMed ID: 24183663

Coelho, D. S., Gaspar, C. J. and Domingos, P. M. (2014). Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila xbp1. PLoS One 9: e105588. PubMed ID: 25136861

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Dourlen, P., Bertin, B., Chatelain, G., Robin, M., Napoletano, F., Roux, M. J. and Mollereau, B. (2012). Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival. PLoS Genet 8: e1002833. PubMed ID: 22844251

Gardner, B. M. and Walter, P. (2011). Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science 333: 1891-1894. PubMed ID: 21852455

Gomez-Navarro, N. and Miller, E. (2016). Protein sorting at the ER-Golgi interface. J Cell Biol 215(6): 769-778. PubMed ID: 27903609

Griciuc, A., Aron, L., Roux, M. J., Klein, R., Giangrande, A. and Ueffing, M. (2010). Inactivation of VCP/ter94 suppresses retinal pathology caused by misfolded rhodopsin in Drosophila. PLoS Genet 6. PubMed ID: 20865169

Han, D., Lerner, A. G., Vande Walle, L., Upton, J. P., Xu, W., Hagen, A., Backes, B. J., Oakes, S. A. and Papa, F. R. (2009). IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates. Cell 138: 562-575. PubMed ID: 19665977

Hollien, J. and Weissman, J. S. (2006). Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response. Science 313: 104-107. PubMed ID: 16825573

Hollien, J., Lin, J. H., Li, H., Stevens, N., Walter, P. and Weissman, J. S. (2009). Regulated Ire1-dependent decay of messenger RNAs in mammalian cells. J Cell Biol 186: 323-331. PubMed ID: 19651891

Kang, M. J. and Ryoo, H. D. (2009). Suppression of retinal degeneration in Drosophila by stimulation of ER-associated degradation. Proc Natl Acad Sci U S A 106: 17043-17048. PubMed ID: 19805114

Lee, A. H., Chu, G. C., Iwakoshi, N. N. and Glimcher, L. H. (2005). XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands. EMBO J 24: 4368-4380. PubMed ID: 16362047

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Li, B. X., Satoh, A. K. and Ready, D. F. (2007). Myosin V, Rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors. J Cell Biol 177: 659-669. PubMed ID: 17517962

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Biological Overview

date revised: 23 June 2023

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