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• W2066604609 abstract "To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3′-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress. To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3′-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress. The ER 3The abbreviations used are: ERendoplasmic reticulummiRNAmicroRNAmiRmicroRNAUPRunfolded protein responseTAP1antigen peptide transporter 1IREinositol-requiring enzymesXBP1spliced XBP1uXBP1unspliced XBP1qRT-PCRquantitative RT-PCRTMtunicamycinALLNN-acetyl-leucinyl-leucinyl-norleucinalCHOPC/EBP homologous proteinC/EBPCAAT/enhancer-binding proteinIFIinterferon-inducedMEFmouse embryonic fibroblast. is the central organelle for the synthesis, folding, and post-translational modification of secretory and membrane proteins. Increased synthesis of secretory pathway proteins and cellular insults that disturb ER homeostasis activate the UPR (1Schröder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2458) Google Scholar, 2Welihinda A.A. Tirasophon W. Kaufman R.J. Gene Expr. 1999; 7: 293-300PubMed Google Scholar). The UPR is primarily a cellular adaptive mechanism that alleviates ER stress by increasing the protein folding capacity and simultaneously reducing the influx of nascent polypeptides into the ER. When stress persists or the recovery mechanisms are ineffective, apoptotic cascades are activated (3Ron D. Walter P. Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4929) Google Scholar, 4Rutkowski D.T. Kaufman R.J. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar, 5Schröder M. Kaufman R.J. Mutat. Res. 2005; 569: 29-63Crossref PubMed Scopus (1413) Google Scholar). UPR-associated cell death contributes to the pathomechanism of a number of human diseases including diabetes mellitus (5Schröder M. Kaufman R.J. Mutat. Res. 2005; 569: 29-63Crossref PubMed Scopus (1413) Google Scholar, 6Fonseca S.G. Gromada J. Urano F. Trends Endocrinol. Metab. 2011; 22: 266-274Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar) and neurodegenerative disorders (7Malhotra J.D. Kaufman R.J. Semin. Cell Dev. Biol. 2007; 18: 716-731Crossref PubMed Scopus (795) Google Scholar, 8Malhotra J.D. Kaufman R.J. Antioxid. Redox Signal. 2007; 9: 2277-2293Crossref PubMed Scopus (1225) Google Scholar). Therefore, delineation of the pathways that govern the adaptive UPR may facilitate development of novel therapies for such diseases. endoplasmic reticulum microRNA microRNA unfolded protein response antigen peptide transporter 1 inositol-requiring enzyme spliced XBP1 unspliced XBP1 quantitative RT-PCR tunicamycin N-acetyl-leucinyl-leucinyl-norleucinal C/EBP homologous protein CAAT/enhancer-binding protein interferon-induced mouse embryonic fibroblast. The mammalian UPR is initiated by three ER transmembrane sensors, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6,) and protein kinase RNA-like ER kinase (PERK) (3Ron D. Walter P. Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4929) Google Scholar). To increase the protein folding capacity of the ER, the transcription factors, IRE1-spliced XBP1 (sXBP1) and ATF6, enhance the expression of ER-resident chaperones and foldases and promote ER expansion (9Bommiasamy H. Back S.H. Fagone P. Lee K. Meshinchi S. Vink E. Sriburi R. Frank M. Jackowski S. Kaufman R.J. Brewer J.W. J. Cell Sci. 2009; 122: 1626-1636Crossref PubMed Scopus (192) Google Scholar, 10Sriburi R. Jackowski S. Mori K. Brewer J.W. J. Cell Biol. 2004; 167: 35-41Crossref PubMed Scopus (522) Google Scholar). To reduce ER protein load during ER stress, the UPR acts at multiple levels. For example, the UPR reduces expression of the cystic fibrosis transmembrane conductance regulator (CFTR) via transcriptional, translational, and post-translational mechanisms (11Bartoszewski R. Rab A. Twitty G. Stevenson L. Fortenberry J. Piotrowski A. Dumanski J.P. Bebok Z. J. Biol. Chem. 2008; 283: 12154-12165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Rab A. Bartoszewski R. Jurkuvenaite A. Wakefield J. Collawn J.F. Bebok Z. Am. J. Physiol. Cell Physiol. 2007; 292: C756-C766Crossref PubMed Scopus (55) Google Scholar). Protein synthesis during the UPR is inhibited through protein kinase RNA-like ER kinase-induced phosphorylation of eIF2α, a modification that leads to a loss of translation initiation complexes (13DuRose J.B. Scheuner D. Kaufman R.J. Rothblum L.I. Niwa M. Mol. Cell Biol. 2009; 29: 4295-4307Crossref PubMed Scopus (80) Google Scholar, 14Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2430) Google Scholar). Additional pathways that reduce ER protein load include IRE1-mediated mRNA cleavage and degradation (15Hollien J. Weissman J.S. Science. 2006; 313: 104-107Crossref PubMed Scopus (925) Google Scholar, 16Hollien J. Lin J.H. Li H. Stevens N. Walter P. Weissman J.S. J. Cell Biol. 2009; 186: 323-331Crossref PubMed Scopus (714) Google Scholar) and ER-associated degradation of proteins that fail to fold properly (1Schröder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2458) Google Scholar, 17Oda Y. Okada T. Yoshida H. Kaufman R.J. Nagata K. Mori K. J. Cell Biol. 2006; 172: 383-393Crossref PubMed Scopus (288) Google Scholar). Because it is clear that the UPR utilizes multiple mechanisms to resolve ER stress, we reasoned that UPR-induced miRNAs may contribute to ER protein and peptide load reduction by decreasing the stability of certain mRNAs (18Guo H. Ingolia N.T. Weissman J.S. Bartel D.P. Nature. 2010; 466: 835-840Crossref PubMed Scopus (3145) Google Scholar). miRNAs are small, ∼22-nucleotide, endogenous RNAs that govern mRNA stability by recruiting the RNA-induced silencing complex to the specific sequences at the 3′-UTR of target mRNAs, resulting in mRNA degradation (18Guo H. Ingolia N.T. Weissman J.S. Bartel D.P. Nature. 2010; 466: 835-840Crossref PubMed Scopus (3145) Google Scholar, 19Bartel D.P. Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (16060) Google Scholar). Because the information is limited regarding ER stress-induced miRNAs and their biological significance, the studies presented herein were designed to select ER stress-induced miRNAs, identify their UPR-associated activators, and characterize their targets. Calu-3 and HeLa cells were obtained from the ATCC. Cells were cultured in DMEM (Invitrogen) with 10% FBS at 37 °C in a humidified incubator at 5% CO2. Glioblastoma multiforme cells and human primary astrocytes were maintained in Dulbecco's modified Eagle's medium plus F12 medium (Invitrogen) supplemented with 10% FBS (HyClone). Cells were split into 6-well plates and allowed to grow until 70–80% confluency prior to experiments. Xbp1+/+ and Xbp1−/− mouse embryonic fibroblasts were provided by Dr. Randal Kaufman, University of Michigan, and were cultured as described previously (9Bommiasamy H. Back S.H. Fagone P. Lee K. Meshinchi S. Vink E. Sriburi R. Frank M. Jackowski S. Kaufman R.J. Brewer J.W. J. Cell Sci. 2009; 122: 1626-1636Crossref PubMed Scopus (192) Google Scholar). Total cellular RNA was isolated using RNeasy (Qiagen). RNA concentration was calculated based on the absorbance at 260 nm. RNA samples were stored at −20 °C. Total cellular RNA + microRNA was isolated using the RNeasy mini kit (Qiagen). RNA samples were stored at −20 °C. The Affymetrix human genome array (HG-U133 Plus 2.0) was used to analyze the mRNA expression pattern of the human airway epithelial cell line Calu-3 (11Bartoszewski R. Rab A. Twitty G. Stevenson L. Fortenberry J. Piotrowski A. Dumanski J.P. Bebok Z. J. Biol. Chem. 2008; 283: 12154-12165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Details about the microarray are available in the supplemental Materials and Methods. The Affymetrix miRNA array was used to analyze the expression pattern of Calu-3 cells. Details about the microarray are available in the supplemental Materials and Methods. We used TaqMan® one-step RT-PCR master mix reagents (Applied Biosystems) as described previously (11Bartoszewski R. Rab A. Twitty G. Stevenson L. Fortenberry J. Piotrowski A. Dumanski J.P. Bebok Z. J. Biol. Chem. 2008; 283: 12154-12165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Rab A. Bartoszewski R. Jurkuvenaite A. Wakefield J. Collawn J.F. Bebok Z. Am. J. Physiol. Cell Physiol. 2007; 292: C756-C766Crossref PubMed Scopus (55) Google Scholar, 20Bartoszewski R. Rab A. Fu L. Bartoszewska S. Collawn J. Bebok Z. Methods Enzymol. 2011; 491: 3-24Crossref PubMed Scopus (37) Google Scholar) using the manufacturer's protocol (57Applied Biosystems Relative Quantification: Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System: Getting Started Guide. Applied Biosystems, Carlsbad, CA2004Google Scholar). Details about the microarray are available in the supplemental Materials and Methods. Real time RT-PCR was performed according to the TaqMan® microRNA and small RNA assays protocol using the TaqMan® microRNA reverse transcription kit (Applied Biosystems). Details about the microarray are available in the supplemental Materials and Methods. To test the impact of hsa-miR-346 on gene expression, we used the miR-346 mimic and miR-346 inhibitor/antagomir (Qiagen). As a control, we used cel-miR-67 (C67, Qiagen), which has no homology to any known mammalian microRNA. HeLa cells grown on 6-well plates were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. ER-TrackerTM Red (glibenclamide BODIPY® TR), anti mouse IgG Alexa Fluor-488, and anti-rabbit IgG AlexaFluor-596 (Molecular Probes) were used according to the manufacturer's protocols. Anti-XBP1 N-terminal polyclonal (rabbit) antibody (Millipore) recognizing both spliced and unspliced XBP1 was used at 1:200 dilution. Anti-TAP1 monoclonal (mouse) antibody (Proteintech Group), anti-KDEL polyclonal (rabbit) antibody (Sigma), and anti-actin polyclonal (rabbit) antibody (Sigma) were used at 1:1000 dilutions. Cells were grown on glass coverslips, washed three times in PBS, and fixed in 4% paraformaldehyde/PBS for 10 min. All incubations were at room temperature unless stated otherwise. Cells were permeabilized with 0.1% Triton X-100/PBS for 5 min, washed three times for 2 min each with PBS, and then blocked with 2.5% goat serum/PBS. Cells were incubated with primary antibody diluted in blocking solution for 2 h. Coverslips were washed five times for 5 min each with PBS. Secondary antibodies were diluted in blocking solution (1:500), and coverslips were incubated for 45 min followed by washing steps and mounted with VECTASHIELD/DAPI (Vector Laboratories). Microscopy was performed using a Leitz epifluorescence microscope equipped with a step motor and a filter wheel assembly (Ludl Electronic Products) and an 83,000-filter set (Chroma Technology). Images were obtained with a SenSys cooled, charge-coupled high-resolution camera (Photometrics). IPLab Spectrum software (Signal Analytics) was used for image acquisition. Cells were lysed in radioimmune precipitation buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl, pH 8.0), supplemented with Complete Mini protease inhibitor (Roche Applied Science) on ice for 15 min. The cell lysates were rotated at 4 °C for 30 min, and the insoluble material was removed by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by BCATM protein assay (Pierce) using bovine serum albumin (BSA) as a standard. Following normalization of protein concentrations, lysates were mixed with equal volumes of 2× Laemmli sample buffer and incubated for 30–45 min at 37 °C prior to separation by SDS-PAGE. Following SDS-PAGE, the proteins from the gel were transferred to polyvinylidene difluoride membranes (300 mA for 90 min at 4 °C). The membranes were then blocked with milk proteins dissolved in PBS/Tween-20 (5% milk, 0.5% Tween 20 for 1–2 h) followed by immunoblotting with the primary antibody specified for each experiment. After the washing steps, the membranes were incubated with HRP-conjugated secondary antibodies (Pierce) and detected using ECL (Pierce). Densitometry was performed using ImageJ (National Institutes of Health). Pharmacological induction of ER stress and activation of the UPR was performed according to previously described methods (11Bartoszewski R. Rab A. Twitty G. Stevenson L. Fortenberry J. Piotrowski A. Dumanski J.P. Bebok Z. J. Biol. Chem. 2008; 283: 12154-12165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Rab A. Bartoszewski R. Jurkuvenaite A. Wakefield J. Collawn J.F. Bebok Z. Am. J. Physiol. Cell Physiol. 2007; 292: C756-C766Crossref PubMed Scopus (55) Google Scholar). Briefly, cells were treated with the compounds for the time periods specified: proteasome inhibition with ALLN (calpain inhibitor I; 100 μm); brefeldin A (0.4 μg/ml, Sigma); 300 nm thapsigargin (Sigma); tunicamycin (5 μg/ml, Sigma); or 4 mm DTT (Sigma) for the time periods specified in each experiment. For the time course experiment, tunicamycin (TM) was added to samples for the time periods specified at 5 μg/ml final concentration (21Lin H.J. Tsai C.H. Tsai F.J. Chen W.C. Chen H.Y. Fan S.S. Mol. Diagn. 2004; 8: 245-252Crossref PubMed Google Scholar). Results were expressed as means ± S.D. Statistical significance among means was determined using the Student's t test (two samples, paired and unpaired). To identify ER stress-induced miRNAs, we utilized human airway epithelial cells, Calu-3, as our primary model. We used two classic compounds, a proteasome inhibitor, ALLN (calpain inhibitor I), and a glycosylation inhibitor, tunicamycin, to induce ER stress (11Bartoszewski R. Rab A. Twitty G. Stevenson L. Fortenberry J. Piotrowski A. Dumanski J.P. Bebok Z. J. Biol. Chem. 2008; 283: 12154-12165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 20Bartoszewski R. Rab A. Fu L. Bartoszewska S. Collawn J. Bebok Z. Methods Enzymol. 2011; 491: 3-24Crossref PubMed Scopus (37) Google Scholar, 22Behrman S. Acosta-Alvear D. Walter P. J. Cell Biol. 2011; 192: 919-927Crossref PubMed Scopus (93) Google Scholar). Isolated RNA samples were first subjected to qRT-PCR to measure the levels of UPR reporters (BiP and spliced XBP1 (sXBP1) mRNA) (12Rab A. Bartoszewski R. Jurkuvenaite A. Wakefield J. Collawn J.F. Bebok Z. Am. J. Physiol. Cell Physiol. 2007; 292: C756-C766Crossref PubMed Scopus (55) Google Scholar) to confirm UPR activation. BiP mRNA levels were increased >14-fold and sXBP1 mRNA levels were increased >3-fold, confirming that the UPR was activated. Next, the samples were subjected to genome-wide miRNA expression arrays followed by bioinformatics analysis. We only selected miRNAs that were increased more than 2-fold by both ER stressors (Fig. 1A). Two miRNAs, miR-346 and miR-885-3p, fit the selection criteria. Based on the most significant increase, we selected miR-346 for further analysis. mir-346 is encoded within the intron 2 of GRID1, also considered a schizophrenia susceptibility gene, on chromosome 10. miR-346 levels are lower in schizophrenic patients, but there is no strong correlation between GRID1 and miR-346 expression, suggesting that miR-346 expression is regulated independently from its host gene (23Zhu Y. Kalbfleisch T. Brennan M.D. Li Y. Schizophr. Res. 2009; 109: 86-89Crossref PubMed Scopus (91) Google Scholar). Subsequent studies demonstrated miR-346 expression in lymphatic (24Thompson L.H. Whiston R.A. Rakhimov Y. Taccioli C. Liu C.G. Croce C. Metcalfe S.M. Cell Cycle. 2010; 9: 4213-4221Crossref PubMed Scopus (9) Google Scholar) and adipose tissues (25Tsai N.P. Lin Y.L. Wei L.N. Biochem. J. 2009; 424: 411-418Crossref PubMed Scopus (119) Google Scholar). Our analysis of GRID1 mRNA levels in the genome-wide mRNA expression arrays and by qRT-PCR indicated that GRID1 mRNA levels did not change during ER stress in Calu-3 or HeLa cells, whereas miR-346 levels were increased under the same conditions (Fig. 1B). Therefore, these results support the view that GRID1 and miR-346 expression are independently regulated. The chromosomal locations of mir-346 and GRID1 are shown in Fig. 1C (based on the GRID1 entry in the GeneCards database). To confirm the predicted increase in miR-346 levels during ER stress and to determine the time period for the induction of miR-346, we performed time course qRT-PCR experiments following ER stress induction with TM. In these experiments, we monitored sXBP1 mRNA as a reporter of UPR activation (12Rab A. Bartoszewski R. Jurkuvenaite A. Wakefield J. Collawn J.F. Bebok Z. Am. J. Physiol. Cell Physiol. 2007; 292: C756-C766Crossref PubMed Scopus (55) Google Scholar) and measured miR-346 levels from the same samples. The results demonstrate that both sXBP1 mRNA and miR-346 levels increase following TM treatment, reaching a maximum at 8 h after ER stress induction (Fig. 1D). These results are consistent with UPR activation (sXBP1 mRNA increase) and simultaneous miR-346 induction, suggesting a UPR-associated transcriptional activation of miR-346 in human airway epithelial cells. To determine whether miR-346 expression can be induced by the UPR in other cell types, we performed studies to measure miR-346 levels in Calu-3, HeLa, primary glioblastoma, and primary astrocytoma cells using a panel of classic ER stressors (supplemental Fig. S1). The results of these studies indicate that miR-346 expression is induced by a common UPR mechanism independent of the ER stressor. Importantly, miR-346 expression increased in response to UPR activation in all cells tested. However, the magnitude of miR-346 induction in response to ER stress varied among cell types, and this may reflect differences in basal expression of miR-346. Because miR-346 expression is enhanced during ER stress without any significant change in GRID1 mRNA, we investigated whether UPR-activated transcription factors can activate miR-346 expression. To determine which transcriptional activator to test, we analyzed the putative regulatory region of the miR-346 gene upstream of the transcription start site using the miRGen 2.0 database (26Alexiou P. Vergoulis T. Gleditzsch M. Prekas G. Dalamagas T. Megraw M. Grosse I. Sellis T. Hatzigeorgiou A.G. Nucleic Acids Res. 2010; 38: D137-D141Crossref PubMed Scopus (123) Google Scholar, 27Landgraf P. Rusu M. Sheridan R. Sewer A. Iovino N. Aravin A. Pfeffer S. Rice A. Kamphorst A.O. Landthaler M. Lin C. Socci N.D. Hermida L. Fulci V. Chiaretti S. Foà R. Schliwka J. Fuchs U. Novosel A. Müller R.U. Schermer B. Bissels U. Inman J. Phan Q. Chien M. Weir D.B. Choksi R. De Vita G. Frezzetti D. Trompeter H.I. Hornung V. Teng G. Hartmann G. Palkovits M. Di Lauro R. Wernet P. Macino G. Rogler C.E. Nagle J.W. Ju J. Papavasiliou F.N. Benzing T. Lichter P. Tam W. Brownstein M.J. Bosio A. Borkhardt A. Russo J.J. Sander C. Zavolan M. Tuschl T. Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3045) Google Scholar). This analysis indicated a binding site for the UPR-activated transcription factor sXBP1 in addition to binding sites for a number of non-UPR specific transcription factors (supplemental Fig. S2). The location of the sXBP-binding site is −1736 upstream of the transcription start site (26Alexiou P. Vergoulis T. Gleditzsch M. Prekas G. Dalamagas T. Megraw M. Grosse I. Sellis T. Hatzigeorgiou A.G. Nucleic Acids Res. 2010; 38: D137-D141Crossref PubMed Scopus (123) Google Scholar, 27Landgraf P. Rusu M. Sheridan R. Sewer A. Iovino N. Aravin A. Pfeffer S. Rice A. Kamphorst A.O. Landthaler M. Lin C. Socci N.D. Hermida L. Fulci V. Chiaretti S. Foà R. Schliwka J. Fuchs U. Novosel A. Müller R.U. Schermer B. Bissels U. Inman J. Phan Q. Chien M. Weir D.B. Choksi R. De Vita G. Frezzetti D. Trompeter H.I. Hornung V. Teng G. Hartmann G. Palkovits M. Di Lauro R. Wernet P. Macino G. Rogler C.E. Nagle J.W. Ju J. Papavasiliou F.N. Benzing T. Lichter P. Tam W. Brownstein M.J. Bosio A. Borkhardt A. Russo J.J. Sander C. Zavolan M. Tuschl T. Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3045) Google Scholar, 28Marson A. Levine S.S. Cole M.F. Frampton G.M. Brambrink T. Johnstone S. Guenther M.G. Johnston W.K. Wernig M. Newman J. Calabrese J.M. Dennis L.M. Volkert T.L. Gupta S. Love J. Hannett N. Sharp P.A. Bartel D.P. Jaenisch R. Young R.A. Cell. 2008; 134: 521-533Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar). Next, we tested whether the overexpression of the transcriptionally active sXBP1 alone would enhance miR-346 expression. Because miR-346 is expressed in HeLa cells (supplemental Fig. S1), these cells were transfected with plasmids expressing sXBP1, unspliced XBP1 (uXBP1), or an empty vector. To ensure the efficiency of the transfection, we measured sXBP1 and uXBP1 mRNA levels and visualized the nuclear localization of sXBP1 and uXBP1 proteins by immunocytochemistry (Fig. 2, A and B). Although uXBP1 is translated and enters the nucleus, it lacks the ability to promote transcription (29Yoshida H. Oku M. Suzuki M. Mori K. J. Cell Biol. 2006; 172: 565-575Crossref PubMed Scopus (316) Google Scholar); therefore, overexpression of this construct served as a specificity control. The results indicate that overexpression of sXBP1 increased miR-346 levels, whereas uXBP1 had no effect (Fig. 2C). Therefore, these studies indicate that sXBP1 overexpression is sufficient for miR-346 transcription. To confirm the role of sXBP1 as transcriptional activator of miR-346 during ER stress, we utilized wild-type (Xbp1+/+) and XBP1 knock-out (Xbp1−/−) mouse embryonic fibroblasts (9Bommiasamy H. Back S.H. Fagone P. Lee K. Meshinchi S. Vink E. Sriburi R. Frank M. Jackowski S. Kaufman R.J. Brewer J.W. J. Cell Sci. 2009; 122: 1626-1636Crossref PubMed Scopus (192) Google Scholar). We induced ER stress with TM for different time periods and then assessed miR-346 expression using mouse-specific primers and qRT-PCR. Although miR-346 expression increased in Xbp1+/+ cells, reaching maximal levels by 8 h, miR-346 levels did not change in Xbp1−/− cells, confirming the role of sXBP1 in miR-346 induction (Fig. 3A). To ensure that the XBP1-independent UPR pathways were intact in the Xbp1−/− cells, we assessed the expression of a known XBP1-dependent and an XBP1-independent gene as controls. ERdj4 is encoded by the XBP1-dependent UPR target gene Dnajb9 (30Adachi Y. Yamamoto K. Okada T. Yoshida H. Harada A. Mori K. Cell Struct. Funct. 2008; 33: 75-89Crossref PubMed Scopus (329) Google Scholar, 31Shaffer A.L. Shapiro-Shelef M. Iwakoshi N.N. Lee A.H. Qian S.B. Zhao H. Yu X. Yang L. Tan B.K. Rosenwald A. Hurt E.M. Petroulakis E. Sonenberg N. Yewdell J.W. Calame K. Glimcher L.H. Staudt L.M. Immunity. 2004; 21: 81-93Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar), and the transcription factor C/EBP homologous protein (CHOP, also known as growth arrest and DNA damage gene 153) is encoded by the XBP1-independent UPR target gene Ddit3 (14Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2430) Google Scholar, 32Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1637) Google Scholar). As expected, ER stress-associated induction of Dnajb9 was markedly compromised in Xbp1−/− cells (Fig. 3B), whereas CHOP expression was strongly induced in Xbp1−/− cells (Fig. 3C). Therefore, these data demonstrate that sXBP1 is essential for induction of miR-346 during ER stress. A model depicting the mechanism of miR-346 induction during ER stress is presented in Fig. 4.FIGURE 4ER stress-associated miR-346 induction. Step 1, induction of miR-346 transcription. ER stress activates IRE1α, which splices the XBP1 mRNA, resulting in the translation of sXBP1 protein. sXBP1 enters the nucleus and activates pri-miR-346 transcription. Step 2, target mRNA degradation. Pri-miR-346 enters the miRNA maturation pathway, and in the cytosol, miR-346 is generated. Mir-346 binds to the 3′-UTR of target transcripts and recruits the RNA-induced silencing complex, leading to mRNA decay.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A previous study indicated that miR-436 targets the 5′-UTR of a mouse brain-specific splice variant of the receptor-interacting protein 140 (RIP140) mRNA and induces its expression (25Tsai N.P. Lin Y.L. Wei L.N. Biochem. J. 2009; 424: 411-418Crossref PubMed Scopus (119) Google Scholar). Based on this, we tested whether RIP140 levels were altered either during ER stress or by miR-346. Our results indicated that neither ER stress nor miR-346 had any effect on RIP140 levels in human Calu-3 airway epithelial cells or in HeLa cells, indicating that RIP140 was not a target. As a more practical method for identifying the ER stress-associated targets of miR-346, we applied miRNA target prediction algorithms such as TargetBoost and TargetScan (33Saetrom O. Snøve Jr., O. Saetrom P. RNA. 2005; 11: 995-1003Crossref PubMed Scopus (102) Google Scholar, 34Saetrom P. Heale B.S. Snøve Jr., O. Aagaard L. Alluin J. Rossi J.J. Nucleic Acids Res. 2007; 35: 2333-2342Crossref PubMed Scopus (259) Google Scholar). These databases were developed based on the observation that the conserved seed pairing sites for miRNAs are often flanked by adenosines (35Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9936) Google Scholar, 36Friedman R.C. Farh K.K. Burge C.B. Bartel D.P. Genome Res. 2009; 19: 92-105Crossref PubMed Scopus (6489) Google Scholar). Because the bioinformatics analysis predicted 2491 potential targets for miR-346, we turned to an experimental approach. We wanted to focus on targets that were repressed by ER stress and contributed to the regulation of ER protein influx. To accomplish this, we overexpressed an miR-346 mimic that resembles mature miR-346, or a control miRNA (miR-C67) that has no mammalian target sequences, in HeLa cells. We then performed mRNA profiling arrays and selected transcripts that were repressed in the miR-346 mimic-expressing cells. This approach identified 28 mRNAs as significantly reduced (>2-fold) when the mature form of miR-346 (mimic) was overexpressed. To select transcripts that were also reduced during ER stress, we cross-referenced the" @default.
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• W2066604609 date "2011-12-01" @default.
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• W2066604609 title "The Unfolded Protein Response (UPR)-activated Transcription Factor X-box-binding Protein 1 (XBP1) Induces MicroRNA-346 Expression That Targets the Human Antigen Peptide Transporter 1 (TAP1) mRNA and Governs Immune Regulatory Genes" @default.
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• W2066604609 doi "https://doi.org/10.1074/jbc.m111.304956" @default.
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