Diabetes Volume 67, October 2018 2019 The No-Go and Nonsense-Mediated RNA Decay Pathways Are Regulated by Inflammatory Cytokines in Insulin-Producing Cells and Human Islets and Determine b-Cell Insulin Biosynthesis and Survival Seyed Mojtaba Ghiasi,1 Nicolai Krogh,2 Björn Tyrberg,3 and Thomas Mandrup-Poulsen1 Diabetes 2018;67:2019–2037 | https://doi.org/10.2337/db18-0073 Stress-related changes in b-cell mRNA levels result from Proinflammatory cytokines have been implied in b-cell a balance between gene transcription and mRNA decay. failure and apoptosis in type 1 and type 2 diabetes (T1D The regulation of RNA decay pathways has not been and T2D) in part by regulation of the b-cell transcriptome investigated in pancreatic b-cells. We found that no-go (1). The levels of ;20% of the .29,000 transcripts in and nonsense-mediated RNA decay pathway compo- human islets are altered by cytokines, including apoptosis- nents (RDPCs) and exoribonuclease complexes were fl and in ammation-related genes (2). Interestingly, 35% of ISLET STUDIES expressed in INS-1 cells and human islets. Pelo, Dcp2, the genes expressed in human islets undergo alternative Dis3L2, Upf2, and Smg1/5/6/7 were upregulated by in- splicing, and cytokines cause substantial changes in the fl ammatory cytokines in INS-1 cells under conditions number of spliced transcripts (2). Most human genes b where central -cell mRNAs were downregulated. These exhibit alternative splicing, but not all alternatively spliced changes in RDPC mRNA or corresponding protein transcripts are translated into functional proteins. This levels were largely confirmed in INS-1 cells and rat/ varies in a cell-specific manner, contributing to tissue human islets. Cytokine-induced upregulation of Pelo, specificity of gene expression (3), and can be modulated Xrn1, Dis3L2, Upf2, and Smg1/6 was reduced by induc- by cellular signals such as those provided by proinflammatory ible nitric oxide synthase inhibition, as were endoplasmic cytokines (4). In pancreatic b-cells, cytokines upregulate reticulum (ER) stress, inhibition of Ins1/2 mRNA, and . accumulated insulin secretion. Reactive oxygen species the expression of 20 genes involved in RNA splicing, and . inhibition or iron chelation did not affect RDPC ex- cytokines induce changes in alternative splicing of 50% fi pression. Pelo or Xrn1 knockdown (KD) aggravated, of the cytokine-modi ed genes as detected by exon array whereas Smg6 KD ameliorated, cytokine-induced INS-1 analysis (4). fi cell death without affecting ER stress; both increased mRNA molecules are prone to damage and delity insulin biosynthesis and medium accumulation but errors, which can cause translation of aberrant mRNA not glucose-stimulated insulin secretion in cytokine- into dysfunctional proteins (5), and so far, an RNA repair exposed INS-1 cells. In conclusion, RDPCs are regu- machinery analogous to that operating to restore damaged lated by inflammatory stress in b-cells. RDPC KD DNA has not been discovered. Rather, cellular RNA sur- improved insulin biosynthesis, likely by preventing veillance mechanisms assure the quality and fidelity of Ins1/2 mRNA clearance. Pelo/Xrn1 KD aggravated, but mRNA molecules (6), resulting in breakdown of damaged Smg6 KD ameliorated, cytokine-mediated b-cell death, RNA. This is generally achieved through targeting aberrant possibly through prevention of proapoptotic and anti- mRNA molecules for degradation by various endogenous apoptotic mRNA degradation, respectively. nucleases. 1Department of Biomedical Sciences, University of Copenhagen, Copenhagen, This article contains Supplementary Data online at http://diabetes Denmark .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0073/-/DC1. 2 Department of Cellular and Molecular Medicine, University of Copenhagen, © 2018 by the American Diabetes Association. Readers may use this article as Copenhagen, Denmark long as the work is properly cited, the use is educational and not for profit, and the 3 Translational Science; Cardiovascular, Renal and Metabolism; and IMED Biotech work is not altered. More information is available at http://www.diabetesjournals Unit, AstraZeneca, Gothenburg, Sweden .org/content/license. Corresponding author: Thomas Mandrup-Poulsen, [email protected]. Received 17 January 2018 and accepted 23 July 2018. 2020 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018 At least four forms of cotranslational mRNA quality Denmark). After 48 h of preincubation, cells were exposed control pathways have been described: nonsense-mediated to cytokine mixture at the concentrations indicated in decay (NMD), Staufen1 (STAU1)-mediated mRNA decay figures or figure legends for the indicated time points, (SMD), no-go decay (NGD), and nonstop decay (NSD) glucolipotoxic (GLT) conditions (0.5 mmol/L palmitate con- (7–9). Both NSD and NGD pathways share the ribosomal jugated with 0.1% albumin + 25 mmol/L glucose), or control dissociation factor Pelo, which is necessary for degradation medium (RPMI-glutamax, 10% FBS, 1% penicillin/strepto- of “non-stop codon” mRNAs and damaged mRNAs that mycin, 50 mmol/L 2-mercaptoethanol). cause translational stalling, respectively (10). In SMD, STAU1 recognizes either stem-loop intramolecular base Human and Rat Islet Culture and Cytokine Exposures . pairing of 39-UTR sequences or intermolecular base pairing Islets from six human heart-beating organ donors ( 80% of 39-UTR sequences with long-noncoding RNA via partially purity, donor characteristics listed in Supplementary Table A complementary Alu elements (8). NMD engaging Upf1, 2, 1 ) were isolated by the European Consortium for Islet 3A, and 3B and Smg1, 5, 6, and 7 degrades mRNAs with Transplantation (ECIT) under local approval in Milan, premature termination codons (PTCs). PTCs can arise in Italy, received in fully anonymous form and precultured cellsthroughvariousmechanisms: germline or somatic muta- as described previously (15). There were no apparent differ- tions in DNA, errors in transcription, posttranscriptional ences in results obtained with islets from male or female mRNA damage, or notably errors in mRNA processing, donors, and data were therefore combined. For experiments, such as alternative splicing (9). Failure to recognize and 50 human islets were cultured in RPMI supplemented with eliminate these mRNA transcripts can result in production 2% human serum (Life Technologies, Naerum, Denmark), of truncated and dysfunctional proteins that directly perturb 1% penicillin/streptomycin, and 5.6 mmol/L glucose for 24 h cell function or accumulate in the endoplasmic reticulum exposed with or without cytokine mixture (300 pg/mL (ER) and thus cause ER stress. rrIL-1b +10ng/mLrhIFN-g +10ng/mLrhTNF-a)or PTCs have been implicated in ;30% of all inherited control medium for 4 days. diseases, indicating that the NMD pathway plays a vital Rat islets were isolated from 1-week-old rat pups and role in survival and health. Expressed sequence tag analysis precultured for 72 h before exposure to cytokines for 12, of RNA-sequencing data of the human transcriptome iden- 18, and 24 h as previously described (16). tified 35% of 5,693 alternatively spliced isoforms to be NMD Single-Cell RNA Sequencing of Pancreatic Islets targets, and at least 12% of human genes have a PTC- The expression of genes in islet cell types was determined containing splice isoform (11), thus necessitating regulated by reanalyzing published human islet single-cell sequenc- unproductive splicing and translation (RUST) to link alter- ing data (donor information in Supplementary Table 1B; native splicing and NMD and regulate the abundance of EBI accession number MTAB-5061) (17). mRNA transcripts (12). Accordingly, NMD inhibition leads to an ;15%increaseinPTC-containingmRNAisoforms(13). Apoptosis and Cell Viability Assays Very little is known about the role of RNA decay in Apoptosis assay was performed in duplicate through de- fl stress regulation of the transcriptome. We therefore hy- tection of either caspase-3 activity using the uorimetric pothesized that cytokines regulate RNA decay with con- caspase-3 activity assay kit (Sigma-Aldrich, Copenhagen, sequences for b-cell survival in response to inflammatory Denmark) or DNA/histone complexes released from the stress. We report that RNA decay pathway components nucleus to the cytosol using the Roche cell death assay kit (RDPCs) are regulated by proinflammatory cytokines in (Roche, Mannheim, Germany) according to the manufac- b-cells in a nitric oxide (NO), but not reactive oxygen turers’ protocols. Cell viability was measured by MTT and species (ROS), dependent manner. RDPC knockdown (KD) Alamarblue assays (Life Technologies), as previously de- partially restored insulin transcripts, content and medium scribed (18,19). accumulation, but not glucose-stimulated insulin secretion NO Assay (GSIS), and modulated cytokine-mediated death in cytokine- As a surrogate of NO production, accumulated nitrite was exposed INS-1 cells. measured in supernatants (100 mL) from the wells used for INS-1 cell apoptosis assay by the Griess reagent, as pre- RESEARCH DESIGN AND METHODS viously described (20). Cell Culture and Cytokine Exposures The rat INS-1 cell line provided by Claes Wollheim (Uni- Real-time Reverse Transcriptase Quantitative PCR versity of Geneva, Geneva, Switzerland) tested negative for Total RNA was extracted using the NucleoSpin kit Mycoplasma and was maintained as previously described (Macheray-Nagel, Bethlehem, PA) according to the man- (14). INS-1 cells were seeded in 6-well plates (1 3 106 ufacturer’s