IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response.

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

ERN1 knockdown modifies the hypoxic regulation of homeobox gene expression in U87MG glioblastoma cells.

OBJECTIVE.: Homeobox genes play an important role in health and disease including oncogenesis. The present investigation aimed to study ERN1-dependent hypoxic regulation of the expression of genes encoding homeobox proteins MEIS (zinc finger E-box binding homeobox 2) and LIM homeobox 1 family, SPAG4 (sperm associated antigen 4) and NKX3-1 (NK3 homeobox 1) in U87MG glioblastoma cells in response to inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioblastoma growth. METHODS.: The expression level of homeobox genes was studied in control (transfected by vector) and ERN1 knockdown U87MG glioblastoma cells under hypoxia induced by dimethyloxalylglycine (0.5 mM for 4 h) by quantitative polymerase chain reaction and normalized to ACTB. RESULTS.: It was found that hypoxia down-regulated the expression level of LHX2, LHX6, MEIS2, and NKX3-1 genes but up-regulated the expression level of MEIS1, LHX1, MEIS3, and SPAG4 genes in control glioblastoma cells. At the same time, ERN1 knockdown of glioblastoma cells significantly modified the sensitivity of all studied genes to a hypoxic condition. Thus, ERN1 knockdown of glioblastoma cells removed the effect of hypoxia on the expression of MEIS1 and LHX1 genes, but increased the sensitivity of MEIS2, LHX2, and LHX6 genes to hypoxia. However, the expression of MEIS3, NKX3-1, and SPAG4 genes had decreased sensitivity to hypoxia in ERN1 knockdown glioblastoma cells. Moreover, more pronounced changes under the conditions of ERN1 inhibition were detected for the pro-oncogenic gene SPAG4. CONCLUSION.: The results of the present study demonstrate that hypoxia affected the expression of homeobox genes MEIS1, MEIS2, MEIS3, LHX1, LHX2, LHX6, SPAG4, and NKX3-1 in U87MG glioblastoma cells in gene-specific manner and that the sensitivity of all studied genes to hypoxia condition is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling, and possibly contributed to the control of glioblastoma growth. A fundamentally new results of this work is the establishment of the fact regarding the dependence of hypoxic regulation of SPAG4 gene expression on ER stress, in particular ERN1, which is associated with suppression of cell proliferation and tumor growth.

Gα12 and endoplasmic reticulum stress-mediated pyroptosis in a single cycle of dextran sulfate-induced mouse colitis.

Inflammatory bowel disease (IBD) pathogenesis involves complex inflammatory events and cell death. Although IBD involves mainly necrosis in the digestive tract, pyroptosis has also been recognized. Nonetheless, the underlying basis is elusive. Gα12/13 overexpression may affect endoplasmic reticulum (ER) stress. This study examined how Gα12/13 and ER stress affect pyroptosis using dextran sulfate sodium (DSS)-induced colitis models. Gα12/13 levels were increased in the distal and proximal colons of mice exposed to a single cycle of DSS, as accompanied by increases of IRE1α, ATF6, and p-PERK. Moreover, Il-6, Il-1ß, Ym1, and Arg1 mRNA levels were increased with caspase-1 and IL-1ß activation, supportive of pyroptosis. In the distal colon, RIPK1/3 levels were enhanced to a greater degree, confirming necroptosis. By contrast, the mice subjected to three cycles of DSS treatments showed decreases of Gα12/13, as accompanied by IRE1α and ATF6 suppression, but increases of RIPK1/3 and c-Cas3. AZ2 treatment, which inhibited Gα12, has an anti-pyroptotic effect against a single cycle of colitis. These results show that a single cycle of DSS-induced colitis may cause ER stress-induced pyroptosis as mediated by Gα12 overexpression in addition to necroptosis, but three cycles model induces only necroptosis, and that AZ2 may have an anti-pyroptotic effect.

The role of the 5' sensing function of ribonuclease E in cyanobacteria.

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.

Endoribonuclease DNE1 Promotes Ethylene Response by Modulating EBF1/2 mRNA Processing in Arabidopsis.

The gaseous phytohormone ethylene plays a crucial role in plant growth, development, and stress responses. In the ethylene signal transduction cascade, the F-box proteins EIN3-BINDING F-BOX 1 (EBF1) and EBF2 are identified as key negative regulators governing ethylene sensitivity. The translation and processing of EBF1/2 mRNAs are tightly controlled, and their 3' untranslated regions (UTRs) are critical in these regulations. However, despite their significance, the exact mechanisms modulating the processing of EBF1/2 mRNAs remain poorly understood. In this work, we identified the gene DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1), which encodes an endoribonuclease and is induced by ethylene treatment, as a positive regulator of ethylene response. The loss of function mutant dne1-2 showed mild ethylene insensitivity, highlighting the importance of DNE1 in ethylene signaling. We also found that DNE1 colocalizes with ETHYLENE INSENSITIVE 2 (EIN2), the core factor manipulating the translation of EBF1/2, and targets the P-body in response to ethylene. Further analysis revealed that DNE1 negatively regulates the abundance of EBF1/2 mRNAs by recognizing and cleaving their 3'UTRs, and it also represses their translation. Moreover, the dne1 mutant displays hypersensitivity to 1,4-dithiothreitol (DTT)-induced ER stress and oxidative stress, indicating the function of DNE1 in stress responses. This study sheds light on the essential role of DNE1 as a modulator of ethylene signaling through regulation of EBF1/2 mRNA processing. Our findings contribute to the understanding of the intricate regulatory process of ethylene signaling and provide insights into the significance of ribonuclease in stress responses.

Endoplasmic reticulum stress responses and epigenetic alterations in arsenic carcinogenesis.

Arsenic is a well-known human carcinogen whose environmental exposure via drinking water, food, and air impacts millions of people across the globe. Various mechanisms of arsenic carcinogenesis have been identified, ranging from damage caused by excessive production of free radicals and epigenetic alterations to the generation of cancer stem cells. A growing body of evidence supports the critical involvement of the endoplasmic stress-activated unfolded protein response (UPR) in promoting as well as suppressing cancer development/progression. Various in vitro and in vivo models have also demonstrated that arsenic induces the UPR via activation of the PERK, IRE1α, and ATF6 proteins. In this review, we discuss the mechanisms of arsenic-induced endoplasmic reticulum stress and the role of each UPR pathway in the various cancer types with a focus on the epigenetic regulation and function of the ATF6 protein. The importance of UPR in arsenic carcinogenesis and cancer stem cells is a relatively new area of research that requires additional investigations via various omics-based and computational tools. These approaches will provide interesting insights into the mechanisms of arsenic-induced cancers for prospective target identification and development of novel anti-cancer therapies.

IRE1 RNase controls CD95-mediated cell death.

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

RACK1 and IRE1 participate in the translational quality control of amyloid precursor protein in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein (APP). Protein quality control systems are dedicated to remove faulty and deleterious proteins to maintain cellular protein homeostasis (proteostasis). Identidying mechanisms underlying APP protein regulation is crucial for understanding AD pathogenesis. However, the factors and associated molecular mechanisms regulating APP protein quality control remain poorly defined. In this study, we show that mutant APP with its mitochondrial-targeting sequence ablated exhibited predominant endoplasmic reticulum (ER) distribution and led to aberrant ER morphology, deficits in locomotor activity, and shortened lifespan. We searched for regulators that could counteract the toxicity caused by the ectopic expression of this mutant APP. Genetic removal of the ribosome-associated quality control (RQC) factor RACK1 resulted in reduced levels of ectopically expressed mutant APP. By contrast, gain of RACK1 function increased mutant APP level. Additionally, overexpression of the ER stress regulator (IRE1) resulted in reduced levels of ectopically expressed mutant APP. Mechanistically, the RQC related ATPase VCP/p97 and the E3 ubiquitin ligase Hrd1 were required for the reduction of mutant APP level by IRE1. These factors also regulated the expression and toxicity of ectopically expressed wild type APP, supporting their relevance to APP biology. Our results reveal functions of RACK1 and IRE1 in regulating the quality control of APP homeostasis and mitigating its pathogenic effects, with implications for the understanding and treatment of AD.

MinD-RNase E interplay controls localization of polar mRNAs in E. coli.

The E. coli transcriptome at the cell's poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown. Here, we combined a candidate system approach with proteomics to identify factors that mediate mRNAs localization to the cell poles. We identified the pole-to-pole oscillating protein MinD as an essential factor regulating polar mRNA localization, although it is not able to bind RNA directly. We demonstrate that RNase E, previously shown to interact with MinD, is required for proper localization of polar mRNAs. Using in silico modeling followed by experimental validation, the membrane-binding site in RNase E was found to mediate binding to MinD. Intriguingly, not only does MinD affect RNase E interaction with the membrane, but it also affects its mode of action and dynamics. Polar accumulation of RNase E in ΔminCDE cells resulted in destabilization and depletion of mRNAs from poles. Finally, we show that mislocalization of polar mRNAs may prevent polar localization of their protein products. Taken together, our findings show that the interplay between MinD and RNase E determines the composition of the polar transcriptome, thus assigning previously unknown roles for both proteins.

Profiling of Unfolded Protein Response Markers and Effect of IRE1α-specific Inhibitor in Pituitary Neuroendocrine Tumor.

CONTEXT: Inositol-requiring enzyme 1α (IRE1α) and PKR-like ER kinase (PERK), which are endoplasmic reticulum (ER) membrane proteins, regulate the unfolded protein response (UPR). These molecules have recently gained attention as a novel therapeutic target in secretory tumors. The roles of the UPR in pituitary neuroendocrine tumors (PitNETs) are unclear. OBJECTIVE: To clarify UPR profiling of PitNETs and to investigate the effect of pharmacological modulation of UPR by KIRA8, a newly developed IRE1α-specific inhibitor. METHODS: In 131 patients with PitNETs, we evaluated RNA expression of UPR markers in PitNETs and their clinical phenotypes. Using GH3 cells, we examined the effects of KIRA8 and its combination with octreotide on UPR profiling, cell growth, and apoptosis. RESULTS: Cytoprotective adaptive-UPR (A-UPR) markers were more increased in functioning PitNETs (FPitNETs, n = 112) than in nonfunctioning PitNETs (NFPitNETs, n = 19), while there was no difference in proapoptotic terminal-UPR (T-UPR) markers. Similarly, overt somatotroph tumors (STs, acromegaly, n = 11) increased A-UPR compared with silent STs (n = 10). In STs, serum IGF-1 levels were inversely correlated with Txnip mRNA expression, a representative T-UPR marker. KIRA8 inhibited cell growth and facilitated apoptosis in GH3 cells with increased expressions of T-UPR markers, which was enhanced by the combination with octreotide. Octreotide increased mRNA expression of Txnip and Chop, but decreased spliced Xbp1 under ER stress. Octreotide is suggested to inhibit activation of IRE1α but to reciprocally induce T-UPR under PERK. CONCLUSION: UPR markers in FPitNETs are implicated as dominant A-UPR but blunted T-UPR. KIRA8, enhanced with octreotide, unbalances the UPR, leading to antitumor effects. Targeting IRE1α may provide a novel strategy to treat PitNETs.

CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis in lung cancer cells via the PERK-ATF4 and IRE1α-JNK pathways.

Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.

Methylparaben induces hepatic glycolipid metabolism disorder by activating the IRE1α-XBP1 signaling pathway in male mice.

Methylparaben (MP), a preservative widely used in daily supplies, exists in both the environment and the human body. However, the potential health risks posed by MP remain unclear. This study aimed to unravel the mechanisms by which MP disrupts glucose and lipid homeostasis. For this, we administered MP to mice and observed changes in glucose and lipid metabolism. MP exposure led to hyperglycemia, hyperlipidemia, visceral organ injury, and hepatic lipid accumulation. RNA sequencing results from mice livers indicated a close association between MP exposure and endoplasmic reticulum (ER) stress, inflammatory response, and glucose and lipid homeostasis. Western blotting and quantitative reverse transcription-polymerase chain reaction revealed that MP activated ER stress, particularly the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway, which further promoted the activation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. The activation of these pathways phosphorylated insulin receptor substrate-1 (IRS1) (ser 307), resulting in decreased phosphorylation of protein kinase B (Akt) (ser 473), leading to insulin resistance. Additionally, MP exposure promoted lipogenesis through ER stress. To explore potential remedies, we administered the ER stress inhibitor 4-phenylbutyric acid (4-PBA) and the IRE1α-XBP1 pathway inhibitor toyocamycin to mice, both of which protected against metabolic disorders and organ injury caused by MP. These findings suggest that MP induces disruptions in glucose and lipid metabolism through ER stress, primarily through the IRE1α-XBP1 pathway.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response.

The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

A conserved protein inhibitor brings under check the activity of RNase E in cyanobacteria.

The bacterial ribonuclease RNase E plays a key role in RNA metabolism. Yet, with a large substrate spectrum and poor substrate specificity, its activity must be well controlled under different conditions. Only a few regulators of RNase E are known, limiting our understanding on posttranscriptional regulatory mechanisms in bacteria. Here we show that, RebA, a protein universally present in cyanobacteria, interacts with RNase E in the cyanobacterium Anabaena PCC 7120. Distinct from those known regulators of RNase E, RebA interacts with the catalytic region of RNase E, and suppresses the cleavage activities of RNase E for all tested substrates. Consistent with the inhibitory function of RebA on RNase E, depletion of RNase E and overproduction of RebA caused formation of elongated cells, whereas the absence of RebA and overproduction of RNase E resulted in a shorter-cell phenotype. We further showed that the morphological changes caused by altered levels of RNase E or RebA are dependent on their physical interaction. The action of RebA represents a new mechanism, potentially conserved in cyanobacteria, for RNase E regulation. Our findings provide insights into the regulation and the function of RNase E, and demonstrate the importance of balanced RNA metabolism in bacteria.

In-vivo screening implicates endoribonuclease Regnase-1 in modulating senescence-associated lysosomal changes.

Accumulation of senescent cells accelerates aging and age-related diseases, whereas preventing this accumulation extends the lifespan in mice. A characteristic of senescent cells is increased staining with ß-galactosidase (ß-gal) ex vivo. Here, we describe a progressive accumulation of ß-gal staining in the model organism C. elegans during aging. We show that distinct pharmacological and genetic interventions targeting the mitochondria and the mTORC1 to the nuclear core complex axis, the non-canonical apoptotic, and lysosomal-autophagy pathways slow the age-dependent accumulation of ß-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1, modulating ß-gal staining via the transcription factor ets-4/SPDEF. We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated ß-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes.

MicroRNA-322 overexpression reduces neural tube defects in diabetic pregnancies.

BACKGROUND: Hyperglycemia from pregestational diabetes mellitus induces neural tube defects in the developing fetus. Folate supplementation is the only effective way to prevent neural tube defects; however, some cases of neural tube defects are resistant to folate. Excess folate has been linked to higher maternal cancer risk and infant allergy. Therefore, additional interventions are needed. Understanding the mechanisms underlying maternal diabetes mellitus-induced neural tube defects can identify potential targets for preventing such defects. Despite not yet being in clinical use, growing evidence suggests that microRNAs are important intermediates in embryonic development and can serve as both biomarkers and drug targets for disease intervention. Our previous studies showed that maternal diabetes mellitus in vivo activates the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the developing embryo and that a high glucose condition in vitro reduces microRNA-322 (miR-322) levels. IRE1α is an RNA endonuclease; however, it is unknown whether IRE1α targets and degrades miR-322 specifically or whether miR-322 degradation leads to neural tube defects via apoptosis. We hypothesize that IRE1α can inhibit miR-322 in maternal diabetes mellitus-induced neural tube defects and that restoring miR-322 expression in developing neuroepithelium ameliorates neural tube defects. OBJECTIVE: This study aimed to identify potential targets for preventing maternal diabetes mellitus-induced neural tube defects and to investigate the roles and relationship of a microRNA and an RNA endonuclease in mouse embryos exposed to maternal diabetes mellitus. STUDY DESIGN: To determine whether miR-322 reduction is necessary for neural tube defect formation in pregnancies complicated by diabetes mellitus, male mice carrying a transgene expressing miR-322 were mated with nondiabetic or diabetic wide-type female mice to generate embryos with or without miR-322 overexpression. At embryonic day 8.5 when the neural tube is not yet closed, embryos were harvested for the assessment of 3 miR-322 transcripts (primary, precursor, and mature miR-322), tumor necrosis factor receptor-associated factor 3 (TRAF3), and neuroepithelium cell survival. Neural tube defect incidences were determined in embryonic day 10.5 embryos when the neural tube should be closed if there is no neural tube defect formation. To identify which miR-322 transcript is affected by maternal diabetes mellitus and high glucose conditions, 3 miR-322 transcripts were assessed in embryos from dams with or without diabetes mellitus and in C17.2 mouse neural stem cells treated with different concentrations of glucose and at different time points. To determine whether the endonuclease IRE1α targets miR-322, small interfering RNA knockdown of IRE1α or overexpression of inositol-requiring transmembrane kinase/endoribonuclease 1α by DNA plasmid transfection was used to determine the effect of IRE1α deficiency or overexpression on miR-322 expression. RNA immunoprecipitation was performed to reveal the direct targets of inositol-requiring transmembrane kinase/endoribonuclease 1α. RESULTS: Maternal diabetes mellitus suppressed miR-322 expression in the developing neuroepithelium. Restoring miR-322 expression in the neuroepithelium blocked maternal diabetes mellitus-induced caspase-3 and caspase-8 cleavage and cell apoptosis, leading to a neural tube defect reduction. Reversal of maternal diabetes mellitus-inhibited miR-322 via transgenic overexpression prevented TRAF3 up-regulation in embryos exposed to maternal diabetes mellitus. Activated IRE1α acted as an endonuclease and degraded precursor miR-322, resulting in mature miR-322 reduction. CONCLUSION: This study supports the crucial role of the IRE1α-microRNA-TRAF3 circuit in the induction of neuroepithelial cell apoptosis and neural tube defect formation in pregnancies complicated by diabetes mellitus and identifies IRE1α and miR-322 as potential targets for preventing maternal diabetes mellitus-induced neural tube defects.

Escherichia coli strains with precise domain deletions in the ribonuclease RNase E can achieve greatly enhanced levels of membrane protein production.

Escherichia coli is one of the most widely utilized hosts for production of recombinant membrane proteins (MPs). Bacterial MP production, however, is usually accompanied by severe toxicity and low-level volumetric accumulation. In previous work, we had discovered that co-expression of RraA, an inhibitor of the RNA-degrading activity of RNase E, can efficiently suppress the cytotoxicity associated with the MP overexpression process and, simultaneously, enhance significantly the cellular accumulation of membrane-incorporated recombinant MPs in bacteria. Based on this, we constructed the specialized MP-producing E. coli strain SuptoxR, which can achieve dramatically enhanced volumetric yields of well-folded recombinant MPs. Ιn the present work, we have investigated whether domain deletions in the E. coli RNase E, which exhibit reduced ribonucleolytic activity, can result in suppressed MP-induced toxicity and enhanced recombinant MP production, in a manner resembling the conditions of rraA overexpression in E. coli SuptoxR. We have found that some strains encoding specific RNase E truncation variants can achieve significantly enhanced levels of recombinant MP production. Among these, we have found a single RNase E variant strain, which can efficiently suppress MP-induced toxicity and achieve greatly enhanced levels of recombinant MP production for proteins of both prokaryotic and eukaryotic origin. Based on its properties, and in analogy to the original SuptoxR strain, we have termed this strain SuptoxRNE22. E. coli SuptoxRNE22 can perform better than commercially available bacterial strains, which are frequently utilized for recombinant MP production. We anticipate that SuptoxRNE22 will become a widely utilized host for recombinant MP production in bacteria.

Emerging role and clinical implication of mRNA scavenger decapping enzyme in colorectal cancer.

BACKGROUND: Turnover of RNA is a regulated process that in part controls gene expression. This process is partly controlled by the scavenger decapping enzyme (DcpS). This study aimed to investigate the expression of DcpS in colorectal cancer (CRC) tissue, to evaluate its prognostic significance in patients with CRC and to investigate potentially targeted genes by DcpS. METHODS: Immunohistochemical analysis was used to determine localization of DcpS in normal and CRC tissue, western blot analysis for quantification of protein expression and qPCR for mRNA expression in normal and CRC tissue and expression in cell lines after silencing using siRNA. Gene array analysis was used to study regulation of genes after silencing of DcpS. Proliferation was studied using BRDU. RESULTS: DcpS expression was localized to the epithelial cells of both control and cancer tissue. Tumor and paired control tissue samples from 100 patients who underwent surgical resection for primary colorectal adenocarcinomas were utilized. mRNA and protein of DcpS was significantly up-regulated in the patients with CRC and the mRNA level was higher in rectal cancer tissue compared to colon cancer tissue (p < 0.05). Lowest tertile levels of DcpS mRNA in cancer tissue was associated with a decreased cancer-specific survival rate with a hazard ratio (HR) of 4.7 (95% CI=1.02-12.3), independent of disease stage. The low level of DcpS mRNA was a predictor of poorer survival in patients with rectal and disseminated cancer and in patients receiving adjuvant treatment (p < 0.05). After silencing DcpS in Caco-2 cancer cells, altered expression of several genes associated with RNA, cell cycle regulation, alternative splicing and microRNA was observed and resulted in 23% increase in proliferation. CONCLUSIONS: These results indicate that DcpS has potential as a prognostic factor for CRC but further studies in a broader cohort are warranted to evaluate the significance of the findings in the clinic.