Proinflammatory Cytokines Suppress Nonsense-Mediated RNA Decay to Impair Regulated Transcript Isoform Processing in Pancreatic β-Cells

Proinflammatory cytokines are implicated in pancreatic β-cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of Nonsense-Mediated RNA Decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in β-cells. A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-βH3 or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. Gain- or loss-of function of two key NMD components UPF3B and UPF2 is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Cyt attenuate NMD activity in insulin-producing cell lines and primary human β-cells. These effects are found to involve ER stress and are associated with downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing, raises or lowers Cyt-induced cell death, respectively, in EndoC-βH3 cells, and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increase alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in extracellular matrix (ECM) including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitises β-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signalling, potentially serving as a protective response against Cyt-induced NMD component expression. Our findings highlight the central importance of RNA turnover in β-cell responses to inflammatory stress.


Introduction
Inflammatory and glucolipotoxic (GLT) stress causing -cell failure and destruction in vitro differentially regulates hundreds of β-cell transcripts (1,2).The upregulation of splicing factors and of proteins involved in pre-mRNA processing gives rise to alternative splicing (AS) events, which in turn deregulates the balance and turnover of transcript isoforms (3).Interestingly, most human mRNAs exhibit alternative splicing, but not all alternatively spliced transcripts are translated into functional proteins and are therefore targeted for degradation via the RNA decay pathways.
In addition to regulating the expression of normal transcripts, the human nonsense-mediated RNA decay (NMD) machinery functions to eliminate premature termination codon (PTC)-containing mRNAs, as reviewed extensively (4).Alternatively, spliced mRNA species and translation of dominant transcript isoforms vary in a cell-specific manner and depends on the capacity of cells to cope with damaged transcripts (5)(6)(7).A substantial number (i.e., around 35%, depending on tissue and physiological conditions) of alternatively spliced variants contain a PTC (4,8,9).Approximately 35% of the cytokines-regulated transcripts in human islets undergo alternative splicing (6), and Cyt profoundly upregulate NMD) in rat and human insulin-producing cell lines and primary β-cells, likely to handle the NMD-load inferred by PTC containing splice variants (4,10,11).
However, in addition to canonical NMD in which all key NMD components function on target transcripts, a second branch of NMD is (in)dependently regulated in an autoregulatory feedback loop by its key factors including UPF2 and UPF3 in a cell type-specific manner as reviewed previously (4,10).
In a previous study (11) we profiled the expressional level of NMD components and their regulation by cytokines and GLT in insulin-producing cells, but the NMD activity and its consequences for the β-cell transcriptome remained to be investigated.Here, using a luciferase-based NMD activity reporter, gain-/loss-of function and RNA-sequencing analyses in rodent and human β-cell systems, we measured NMD activity and explored its consequences for function and viability of pancreatic β-cells under normal conditions and inflammatory stress.

Materials and Methods
Cell culture, human islet dispersion and treatment INS1(832/13) (12), EndoC-βH3 (13) or dispersed human islet cells were cultured and manipulated according to the protocols and procedures described in Supplementary Methods.

Luciferase-based NMD activity assay
One million cells were co-transfected with 650 ng of plasmid encoding either human Haemoglobin-β (HBB) wildtype (WT or PTC-) or with a PTC-containing mutation (NS39 or PTC+) fused with Renilla (RLuc), in brief named PTC-and PTC+), respectively.Firefly (FLuc) plasmid ( 14) was used as transfection efficiency reference.Renilla and Firefly luminescence was measured by Dual-Luciferase Reporter Assay (Promega, Hampshire, England) (Supplementary Methods).RLuc signals were normalized to the FLuc control in both HBB NS39, in the following named HBB(PTC+) and HBBWT, in the following named HBB (PTC-), and NMD activity was calculated by dividing the RLuc/FLuc-HBB PTC-) by the RLuc/FLuc-HBB PTC+) (Supplementary Fig. 1A) (14).Experiments where the control construct RLuc/FLuc-HBB(PTC-) was affected by cytokines were excluded, so that the resulting NMD activity only denotes the PTC-containing HBB( PTC+).The transfection efficiency was tested twice and resulted in an average of 80% in INS1 and EndoC-βH3 cells as measured by FACS analysis of cells transfected with a GFP expressing plasmid (Supplementary Fig. 1B-C).

Functional analysis of UPF3A/B overexpression
One million INS1(832/13) or EndoC-βH3 cells were transfected with 650 ng of plasmids encoding UPF3A, UPF3B or UPF3BΔ42 (15), then simultaneously with NMD activity reporter plasmids (as above, 650 ng/million cells), recounted and seeded for Western blotting, glucose-stimulated insulin secretion (GSIS), viability, apoptosis (detailed below) and NMD activity assays in relevant plates and pre-incubated for 48 h before treatment with cytokines as explained in the Supplementary Methods.

Lentiviral shRNA gene knock-down
GPIZ lentiviral shRNAs particles directed against UPF2, Upf3A or Upf3B, and a non-silencing shRNA (NS) as negative control, were produced using the Trans-Lentiviral shRNA Packaging System in HEK293 cells (Horizon, Cambridge, England) according to the manufacturer's protocol (Supplementary Methods).

Library preparation, RNA-sequencing, and data analysis
Thirty-three independent biological replicates of total RNA from the NS control and or UPF2 KD EndoC-βH3 cells exposed to cytokines, GLT, or PBS (i.e., N=6 of each PBS-/or cytokines-exposed NS control and UPF2 KD, and N=4/N=5 of GLT-exposed NS control/UPF2 KD, respectively) was extracted using TRIZOL, treated with DNase, and precipitated with isopropanol (Supplementary Methods).One µg total RNA/per isolate was used as input for generation of sequencing libraries using NEBNext®Ultra-TM RNA Library-Prep (Cat#E7770, NEB, Ipswich, USA) following manufacturer's recommendations (Supplementary Methods).The RNA-seq raw data underwent quality control were mapped to human reference genome (16) and analysed using the bioinformatic pipeline described in the Supplementary Methods.
The raw data was analysed through -∆Ct as described in Supplementary Methods.

Statistical analysis
Data are presented as means ± SEM.Statistical analysis was carried out on raw data also in cases where figures give normalized data.Group comparisons were carried out by two-or one-way ANOVA as appropriate.Significant ANOVAs were followed by post-hoc paired Student's t-test with Bonferronicorrection using GraphPad Prism 6.0 (La Jolla, USA).Paired t-test was chosen to normalize for interpassage variability in outcome parameters.Since the experimental conditions did not allow sequential sampling from the same cell culture, parallel control and interventional plate wells were considered to be paired observations and analysed accordingly statistically.If the post-hoc paired t-test did not reveal a carrying statistical difference by ANOVA, individual paired t-tests were performed and corrected for multiple comparisons.Bonferroni-corrected P-values ≤0.05 were considered significant and ≤0.10 a trend.
We next examined whether cytokines-mediated suppression of NMD was consistent with an accumulation of HBB(PTC+) transcripts.For this, we used a forward and reverse primer set to amplify the Renilla gene and the junction of exons 1 and 2 (i.e., ensuring amplification of mature transcripts only), respectively.RT-qPCR analysis demonstrated that cytokines caused significant up-regulation of HBB(PTC+), but not HBB(PTC-) mRNA levels, rendering a significant reduction of the relative PTC-/PTC+ mRNA levels in INS1(832/13) (p=0.008)(Supplementary Fig. 2A-v and vi) and EndoC-βH3 (p=0.001) cells (Fig. 1A-ii; Supplementary Fig. 2B-iv), which verified the suppressive effect of cytokines on NMD activity.
We also examined the effect of 25 mM glucose or GLT conditions on the NMD activity.Unfortunately, these conditions significantly affected the luciferase signal (RLuc/FLuc) from the HBB(PTC-) control, preventing further study of the effects of metabolic stressors on NMD activity.
Taken together, these results show that cytokines suppress the activity of the NMD activity in a range of insulin secreting cell types.

Cytokines-induced suppression of NMD activity in β-cells is ER stress dependent
Whereas NMD degrades unfolded protein response (UPR)-induced transcripts in compensated ER stress, NMD is suppressed in response to pronounced endoplasmic reticulum (ER) stress to allow a fullblown UPR (19,20).Cytokines induce a robust ER stress in pancreatic β-cells, largely via nuclear factor-κB (NF-κB) activation and production of nitroxidative species that inhibit the smooth endoplasmic reticulum Ca 2+ ATPase (SERCA) 2B pump, leading to ER calcium depletion (11,17).We have previously shown that chemical inhibition of inducible nitric oxide synthase (iNOS) alleviated ER stress and normalised cytokines-mediated regulation of NMD components in INS1 cells (11).Therefore, we asked, if cytokines-mediated reduction of NMD activity was dependent on an ER stress response in cells.We first demonstrated that thapsigargin (TG), a non-competitive inhibitor of SERCA (21) and ER stress inducer (22) inhibited NMD activity by 50% in EndoC-βH3 cells as measured by luciferase assay (Supplementary Fig. 3A-i and ii).Compared to untreated EndoC-βH3 cells (Unt), cytokines significantly augmented the increase in mRNA levels encoding the ER stress markers BiP, Xbp1 and Chop (FDR <0.05) measured by RNA-sequencing analysis (Fig. 2A-i), and later verified by RT-qPCR examination (Fig. 2A-ii-vi).Finally, compared with Unt, cytokines significantly decreased the NMD activity by 30%, and this effect was counteracted by the protein kinase R-like endoplasmic reticulum kinase (PERK) phosphorylation inhibitor GSK157 (8 µM) and by the Inositol-Requiring Enzyme1 (IRE1α) endoribonuclease inhibitor 4µ8C (16 µM) in EndoC-βH3 cells (Fig. 2B-i, Supplementary Fig. 3B and Supplementary Fig. 3C-i and ii).RT-qPCR analysis of the relative PTC-/PTC+ mRNA levels in EndoC-βH3 cells confirmed the NMD activity data (Fig. 2B-ii, Supplementary Fig. 3C-iii).
Taken together, these results demonstrate that inhibition of UPR antagonises the cytokine-mediated reduction of NMD activity in EndoC-βH3, indicating that cytokine-mediated inhibition of NMD activity is UPR-dependent.

Cytokines-induced suppression of NMD activity is associated with UPF3B downregulation and attenuated by UPF3 overexpression in β-cells
Since we observed in our previous study that cytokines-induced ER stress downregulated UPF3B expression in human and rodent β-cells, as recovering nitroxidative-driven ER stress using the inducible nitric oxide synthase (iNOS) inhibitor N-methyl-l-arginine (NMA) (11), since transcripts encoding UPR components are NMD targets and have been shown to be stabilised by UPF3A/B depletion (19) and since UPF3B is a NMD activator in mammalian cells (23), which led to the proposal of Upf3-dependent and -independent branches of the NMD pathway (4,10,24).we reasoned that UPF3 regulated NMD activity in -cells.We therefore first measured the UPF3A/B expression level and next investigated the functional impact of overexpressing UPF3A/B on cytokines-mediated suppression of NMD activity in β-cells.RT-qPCR examination showed that cytokines significantly downregulated UPF3B mRNA levels after 18 h in both EndoC-βH3 (Fig. 3A-i and ii) and INS1(832/13) (Supplementary Fig. 4A-i and ii) as previously reported (11).Immunoblot analysis verified overexpression of UPF3A, UPF3B and the UPF3B dominant negative UPF3BΔ42 in both EndoC-βH3 (Supplementary Fig. 4B-i) and INS1(832/13) (Supplementary Fig. 4C-i).Cytokines reduced NMD activity and overexpression of UPF3B significantly attenuated this reduction in EndoC-βH3 (Fig. 3B and Supplementary Fig. 4B-ii) and to a lesser extent in INS1(832/13) (Supplementary Fig. 4C-ii and iii).Neither UPF3A nor UPF3BΔ42 overexpression counteracted cytokines-attenuated NMD activity.This result suggests that cytokines reduce the NMD activity in β-cells through downregulation of UPF3B expression.

UPF3 overexpression deteriorates cell viability and reduces insulin content, but not secretion in EndoC-βH3 cells
The above findings provide evidence that the UPF3-dependent branch of NMD is involved in cytokinesmediated suppression of NMD activity.Therefore, we next investigated the impact of UPF3A/B overexpression on cytokines-induced cell death and insulin secretion.While UPF3A or UPF3B overexpression increased basal cell death, it also exacerbated the cytokines-induced apoptosis in EndoC-βH3 cells (Fig. 4A-i and ii).In INS1(832/13) cells neither UPF3A nor UPF3B overexpression changed cell viability in the absence of Cyt exposure, but UPF3B over-expression significantly aggravated cytokinesinduced cell death as measured by Alamarblue and caspase-3 activity assays (Supplementary Fig. 5A-i and ii).Therefore, we next explored the impact of UPF3A or UPF3B deficiency on β-cell viability.
Lentiviral shRNA-mediated knockdown of UPF3A and or UPF3B (Supplementary Fig. 5B-i and ii) significantly reduced basal INS1(832/13) cell viability (Supplementary Fig. 5C-i-iv).Taken together, this data indicates that genetic manipulations of UPF3A/B could be possibly detrimental for the β-cell viability.
Nonetheless, UPF3B overexpression profoundly lowered insulin content in EndoC-βH3 cells (Fig. 4Biii).In contrast, knockdown of UPF3A or UPF3B significantly decreased the stimulatory index, as well as provoking a substantial increase in insulin content in control INS1(832/13) cells (Supplementary Fig. 5G-ii and iii, and Supplementary Fig. 5H-ii and iii), without altering ins1 or 2 mRNA expression (Supplementary Fig. 5 G-iv and v, Supplementary Fig. 5 H iv and v).
Taken together, these findings reveal that UPF3 overexpression induces basal cell death and exacerbates cytokines-mediated toxicity in β-cells.

UPF2 knockdown potentiates cytokines suppression of NMD activity and slightly alleviates cytokines toxicity for cell viability and insulin content in EndoC-βH3 cells
Next, we investigated the effect of UPF2 deficiency on the viability and insulin secretion of β-cells because (i) UPF3A and UPF3B are involved in regulating UPF2, a key core NMD activator in mammalian cells (25,26) by sequestering away from and bridging the exon-junction complex (EJC) with UPF1 and UPF2, respectively, leading to the NMD activation (23), and genome-wide association data (GWAS) data reveal that the UPF2 variant rs145580445 is significantly associated with type 2 diabetes risk (7).We therefore knocked down the UPF2 gene in EndoC-βH3 cells using RNA interference and chose the three cell lines in which UPF2 was most efficiently knocked down (KD) (Supplementary Fig. 6A-i and ii).Examination of NMD activity using the luciferase-based NMD reporter revealed that UPF2 KD profoundly reduced NMD activity in untreated and cytokines-treated EndoC-βH3 cells (Supplementary Fig. 6B-i and ii).Compared with NS control, UPF2 KD slightly, but significantly prevented cytokines-induced cell death (Fig. 5A-i and ii).UPF2 KD had no effect on the GSIS, but significantly increased insulin content (Fig. 5B-i, ii and iii).
These data indicate that the UPF2 plays a crucial role in cytokines-induced β-cell apoptosis.In addition, the increase in insulin content in UPF2 deficient EndoCβH3 cells implies that insulin transcripts could possibly be targets of the UPF2-dependent NMD pathway branch.

UPF2 knockdown differentially affects cytokines-and glucolipotoxicity-mediated deregulation of EndoC-βH3 transcripts
Consistent with our observations above (Fig. 5), we previously reported (11) that the deficiency of SMG6, an endoribonuclease and a key effector of NMD, rendered protection against cytokines-induced cell death and was associated with increased insulin content.Therefore, we aimed to identify potential NMD target transcripts by using RNA-sequencing to assess the transcriptome of cytokines-or PBStreated EndoC-βH3 cells stably transfected with a non-silencing shRNA (NS) or the specific shRNA (shRNA-1 named U1) against UPF2.Due to the differential effects of Cyt and GLT on UPF3 expression cf.above, we also performed RNA-sequencing after UPF2 KD vs. NS control EndoC-βH3 exposed to GLT compared with PBS-treated.
The RNA-seq datasets from either UPF2 KD or NS control EndoC-βH3 cell lines exposed to cytokines and or GLT was dimensionally reduced by principal component analysis (PCA) into two main principal components, PC1 and PC2 (p<0.05).The PCA of the NS control EndoC βH3 cells demonstrated a high similarity between the biological replicates, a small within-group variance, and a distinct clustering of the untreated, cytokines and GLT groups (Supplementary Fig. 7A-i and ii).Pearson correlation (p<0.05) between samples justified the clustering of biological replicates of cytokines, GLT and untreated conditions (Supplementary Fig. 7B-i and ii).PCA revealed that the UPF2 knockdown increased the majority of variance in transcript isoforms of the cell, hence patterns leading to visually dispersed biological replicates from the cytokines-exposed isolates, whereas decreased the variances from the GLT and untreated biological replicates, hence clustered them together.
To identify UPF2 KD-regulated transcripts possibly providing protection against cytokines-induced cytotoxicity, we interrogated cytokines-and/or GLT-regulated transcripts that were differentially expressed (p<0.05) in UPF2 KD versus NS control EndoC-βH3 cells using gene enrichment analysis (GEA).Gene Ontontolgy (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) gene enrichment analyses demonstrated that GLT significantly (p<0.05)regulated transcripts in the cellular functions of RNA splicing, mitochondrial inner membrane, and purine nucleoside metabolism (Supplementary Fig. 7E-i and ii), although these were not affected by UPF2 KD (Fig. 6B-i).In contrast, Gene Enrichment Analysis (GEA) revealed that in both untreated and cytokines-treated EndoC-βH3 cells, UPF2 KD significantly regulated transcripts encoding proteins involved in synaptic transmission, extracellular matrix, basolateral plasma membrane, receptor complex, synaptic membrane, transcriptional repressor activity and enzyme inhibitor activity (Fig. 6B-iii; Fig. 6C; Supplementary Fig. 7E-iii and iv).
RT-qPCR confirmed the logarithmic fold change of UPF2 KD regulated transcripts in the cytokinestreated EndoC-βH3 cells versus cytokines-treated NS control cells (Fig. 6D).The role of many of these transcripts in cell viability or insulin secretion was previously identified in pancreatic β-cells.Among them, -1-antitrypsin (abbreviated 1-AT) has been proposed as an antagonist against cytokines-induced pancreatic β-cell death (27,28).Our expression profiling verified that cytokines upregulated -1antitrypsin, and this effect was further potentiated by UPF2 KD.To explore the potential importance of these changes, we knocked down -1-antitrypsin using specific siRNAs in INS1(832/13) and EndoC-βH3 cells as confirmed by quantitative WB (Supplementary Fig. 8-i, ii and iii).The effect of siRNAmediated -1-antitrypsin knockdown was inconclusive in EndoC-βH3 cells (Supplementary Fig. 7B-i and ii).Both -1-antitrypsin siRNAs aggravated cytokine-induced cell death in comparison with NS control in INS1(832/13) cells (Fig. 7A-iii and iv).Compared with NS control, -1-antitrypsin knockdown reduced GSIS index, but had no effect on insulin content in INS1(832/13) cells (Fig. 7B-ii, and iii), possibly due to the reduced cell number.
Cytokines reportedly upregulate >30 splicing factors, affecting alternative splicing of 35% of genes in the human islet transcriptome (6).We examined RNA-seq datasets for alternative splicing (AS) isoforms (Supplementary Fig. 9 i) driven by cytokines or GLT versus untreated in the NS control and UPF2 KD EndoC-βH3 cells.Among 2123 and 2106 cytokines-driven AS isoforms, skipped exon (SE) isoforms constituted 70.89% (p=0.1, n=6) and 72.5% (p=0.1, n=6) in NS control and UPF2 KD cells, respectively (Supplementary Fig. 9-ii and iii).In contrast, 220 and 133 GLT-driven AS isoforms were identified in NS control and UPF2 KD, respectively (Supplementary Fig. 9-iv and v).This differential regulation could possibly provide a reliable measure for cytokines and GLT role in inducing AS isoforms in βcells.
Taken together, the above transcriptome analysis of EndoC-βH3 cells indicates that cytokines increase − expression, and this is synergised by NMD attenuation.

Discussion
In this study, we demonstrate that cytokines decrease nonsense-mediated RNA decay (NMD) in INS1(832/13), EndoC-βH3 cells and dispersed human islets.We also showed that the cytokinesmediated decrease of NMD activity was driven by ER stress and downregulation of UPF3B.Loss-/or gain-of function of NMD activity could be elicited by UPF3B over-expression or UPF2 knockdown, which led to increases in, or slight decreases in, cytokines-induced apoptosis associated with decreased and increased insulin contents, respectively, without affecting GSIS index in EndoC-βH3 cells.
Transcriptome profiling indicated a potentiating effect of UPF2 knockdown on Cyt, but not GLTmediated, NMD activity.Interestingly, this approach identified transcript targets encoding proteins belonging to the extracellular matrix such as -1-antitrypsin.Importantly, the knockdown of this gene enhanced cytokines-induced cytotoxicity in β-cells.
To the best of our knowledge the present study represents the first demonstration of a functional effect of cytokines on NMD activity.UPR activation is known to inhibit NMD via PERK activation and eIF2 phosphorylation to restore IRE1α accumulation and hence a robust UPR activation (19,20,29); additional to the role of PERK activation, our findings suggest that IRE1α riboendonuclease activity (p=0.1) was involved in cytokinesmediated NMD inhibition in EndoC-βH3 cells.
UPF3A and UPF3B act as a potent NMD inhibitor and activator, respectively, in HeLa cells and in mice (23), consistent with our observations following UPF3B overexpression in  cells.However, the finding that forced UPF3A overexpression slightly increased NMD activity in β-cells seems inconsistent with previous findings.Recent studies (30,31) support our apparently discrepant finding regarding the effects of UPF3A overexpression by showing redundancy of UPF3A and UPF3B as modular activators of NMD (24).With these two earlier studies in mind, we cannot rule out the interference of endogenous UPF3A in the actions of UPF3B on NMD in β-cells.
We investigated the consequences of NMD activity for pancreatic β-cell function and viability.The increase of NMD activity by UPF3B overexpression induced basal and cytokines-induced cell death in EndoC-βH3 cells, highlighting the role of increased UPF3B level in β-cells.This appears to be relevant for β-cell viability in both normal and inflammatory stress conditions.Similarly, UPF3B knockdown also caused basal cell death in INS1 cells.Hence, basal UPF3A/B levels seem to play crucial roles in the cell viability of β-cells and perturbation of such a controlled level implicates in cell death.On the other hand, the slight protection against cytokines-induced cell death conferred by UPF2 knockdown in EndoC-βH3 cells (Fig. 5A-i and ii) and by SMG6 knockdown in INS1 cells (11) implies a possibly protective mechanism against cytotoxicity of cytokines in β-cells, irrespective to the outcome cytokinesinduced cell death.Moreover, our findings provide evidence that the reduced insulin content observed after UPF3B overexpression is related to overactivated NMD.
Cytokines-induced perturbation of NMD, (potentiated by UPF2 silencing), might change the balance of anti-/pro-apoptotic transcripts.This, in turn, may contribute to cytotoxic damage.Consistent with this view, GEA revealed that cytokines deregulate transcripts encoding proteins that localise to and/or function in the extracellular matrix Thus, -1-antitrypsin knockdown increased the detachment of MIN6 cells and exacerbated Thapsigargin-induced cell death as measured by Propidium-Iodide staining (28) and in this study increased cytokines-induced cell death in INS1(832/13) cells associated with decreased GSIS index.
We speculate that the perturbation of NMD by cytokines leads to increased exon skipping and that this may be part of a feedback loop promoting β-cell plasticity and resilience against cytotoxic cytokines.Future studies will be needed to test this possibility.We note also that depletion of alternative splicing factors (reviewed in (3)) inhibits insulin secretion and induces basal apoptosis and after treatment with cytokines in rodent and human β-cells (3,7,32,33).Moreover, antisense-mediated exon skipping of 48-50 exons of the dystrophin gene restores the open reading frame and allows the generation of partially to largely functional protein (34).
In conclusion, we reveal that cytokines suppress NMD activity via ER stress signalling, possibly as a protective response against cytokines-induced NMD component expression.Our findings highlight the central importance of RNA turnover in β-cell responses to inflammatory stress.
Limitations and Future Perspectives.We used a luciferase-based NMD reporter based on two separate PTC-and PTC+ constructs whose labelled luciferase is separately measured.Thus, a yet-to develop NMD activity reporter by which transcripts RNA, protein, or their corresponding labelled luciferase activity of both PTC-and PTC+ transcripts could be examined in one cell rather than (potentially) two separate cells will remove the limitation of current reporter based on transfection of the constructs into two separate cells.Moreover, the constant overexpression of UPF3A and UPF3B may result in cell death as β-cells cannot cope with overwhelming level of these proteins above the basal level.This could explain why UPF3B overexpression reduced the basal cell viability.Further studies should: 1) conduct in vivo experiments to validate the findings observed in vitro and determine if the regulation of NMD in pancreatic ß-cells is consistent across different cell types and conditions, 2) investigate the mechanisms underlying the regulation of NMD in pancreatic β-cells, including the role of specific NMD components and the impact of different stressors on NMD activity, 3) explore the potential therapeutic implications .CC-BY 4.0 International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 28, 2024.; https://doi.org/10.1101/2023.12.20.572623doi: bioRxiv preprint Regulation of nonsense-mediated decay in pancreatic β-cells of targeting NMD in the treatment of inflammatory stress in β-cells, including the development of novel drugs or therapies that modulate NMD activity.

Translatability of the findings
The findings report NMD involvement in the development of islet autoimmunity and the destruction of pancreatic β-cells in type 1 diabetes as well as islet inflammation in type 2 diabetes.The identification of novel targets arisen from cytokines-driven NMD attenuation could possibly suggest new biomarkers to monitor disease progression and may also guide the development of protein-based vaccines or antisense mRNA therapeutics in individuals who are at risk of diabetes development and or other inflammatory and autoimmune disorders.A-B.EndoC-βH3 cells (A) and dispersed human islet cells (B) were co-transfected with Renilla-HBB(WTnamed PTC-) and or Renilla-HBB(NS39 (named PTC+), and the Firefly plasmids and exposed to cytokines combination and or PBS as untreated (Unt) simultaneously with or without Cycloheximide (CHX) as positive control for inhibited NMD activity.Luciferase activity was measured in the lysate of the transfected EndoC-βH3 cells (A-i) and dispersed human islet cells (B) exposed to cytokines combination (Cyt; 3 ng/mL IL-1β + 10 ng/mL IFNγ+ 10 ng/mL TNFα) for 18 hrs.A-ii.mRNA level of Renilla-HBB fused gene and Firefly gene in the transfected EndoC-βH3 cells was quantified by RT-qPCR using specific primers extending the junction of exons 1 and 2 of the HBB gene, and Renilla gene, or only Firefly gene and normalised to actin and tubulin, respectively.The data are means ± SEM of N=6.The symbol * indicates the Bonferroni-corrected paired t-test values of treatments versus untreated (Unt) (A-B) or otherwise, cytokines (Cyt) that is designated by a line on top of the bars (A-B).* ≤ 0.05, ** ≤ 0.01, *** ≤0.001, **** ≤ 0.0001.ns: non-significant.HBB: Haemoglobin-β, PTC: premature termination codon.On the top of Fig. 1A-i, RLuc: Renilla Luciferase and Ex: exon.

Figure 2. Cytokines-induced suppression of NMD activity in β-cells is ER stress dependent
A. mRNA levels of ER stress markers in EndoC-βH3 cells exposed to cytokines combination (Cyt; 3 ng/mL IL-1β + 10 ng/mL IFNγ+ 10 ng/mL TNFα) for 18 hrs was quantified by RNA-sequencing (A-i) with false discovery rate (FDR) <0.05 presented as logarithmic fold change the cytokines (Cyt) treatment versus control (untreated), and RT-qPCR (A-ii, iii, iv and v) which was normalised to tubulin mRNA.B. EndoC-βH3 cells were co-transfected with Renilla-HBB (PTC-) and or Renilla-HBB (PTC+), and the Firefly plasmid and exposed to PBS as untreated (Unt), cytokines combination (Cyt; 3 ng/mL IL-1β + 10 ng/mL IFNγ+ 10 ng/mL TNFα) alone, and or simultaneously with 16 µM of 4μ8C, an endoribonuclease inhibitor of IRE1α, and or 8 µM of GSK2656157 (GSK157), PERK inhibitor for 18 hrs.B-i.Luciferase activity was measured in the lysate of EndoC-βH3 cells transfected with Renilla-HBB(PTC-) and or Renilla-HBB(PTC+), and the Firefly plasmid exposed to PBS as untreated (Unt) or given conditions, and represented as NMD activity calculated by dividing luciferase activity of HBB(PTC-)/HBB(PTC+) as explained in the methods.B-ii.mRNA level of Renilla-HBB fused gene and Firefly gene in the EndoC-βH3 cells was quantified by RT-qPCR using specific primers extending the junction of exons 1 and 2 of the HBB gene, and Renilla gene, or only Firefly gene and normalised to tubulin.

UPF3B downregulation and attenuated by UPF3 overexpression in β-cells
EndoC-βH3 cells were co-transfected with empty vector (E.V.), UPF3A, UPF3B and or UPF3BΔ42 (dominant negative of UPF3B) plasmids, and then with Renilla-HBB(PTC-) and or Renilla-HBB(PTC+), along with the Firefly plasmid and exposed to cytokines combination (Cyt; 3 ng/mL IL-1β + 10 ng/mL IFN-γ+ 10 ng/mL TNFα) for 18 hrs.A-i and ii.mRNA level of Upf3A and Upf3B genes in EndoC-βH3 cells was quantified by RT-qPCR and normalised to tubulin mRNAs.B. Luciferase activity was measured in the lysate of the transfected cells and represented as NMD activity calculated by dividing luciferase activity of HBB(PTC-)/HBB(PTC+) as explained in the methods.The overexpression of UPF3A and UPF3B proteins was examined by Western blot analysis (Supplementary Fig. 4Bi
A. Volcano plot of number of transcripts (FDR <0.05) regulated by Cyt or GLT versus untreated in the NS or UPF2 KD (U1) cell lines.B. Top enriched pathways (subpanels i and iii) and the number of transcripts (subpanels ii and iv) regulated by Cyt and/or GLT in the UPF2 KD (FDR < 0.0.5).Enrichment is shown as log10 (adjusted p-value <0.05).C. Top GEA-identified transcripts regulated by Cyt compared to untreated in UPF2 KD vs. NS control EndoC-βH3 cells.The expression level is shown as log2 (adjusted p-value <0.05).D. RT-qPCR verification of the identified transcripts regulated by Cyt in UPF2 KD vs. NS control EndoC-βH3 cells.The expression level is shown as log2 (adjusted p-value <0.05).EndoC-βH3 and INS1(832/13) cells were transfected with siRNAs against SERPINA1 (two species-specific siRNAs for each cell type; si1 and si2) and a non-silencing siRNA control (NS), incubated for 24 hours and exposed to PBS as untreated (Unt) and cytokines combination (Cyt for EndoC-βH3; 3 ng/mL IL-1β + 10 ng/mL IFN-γ+ 10 ng/mL TNFα) (Cyt for INS1(832/13); 150 pg/mL IL-1β + 0.1 ng/mL IFNγ+ 0.1 ng/mL TNFα) for 72 and 24 hrs, respectively (see Supplementary Methods).The knockdown efficiency was checked using quantitative WB (Supplementary Fig. 8-i, ii and iii). A. Cell viability was measured by Alamarblue (A-i and iii) and caspase-3 activity (A-ii and iv) assays (N=6).B. Glucose-stimulated insulin secretion (GSIS) (C-i) and insulin contents (C-iii) were investigated in the transfected EndoC-βH3 cells.Insulin concentration (ng/ml) was measured by insulin ultra-sensitive assay (N=6).GSIS index (C-ii) was calculated by dividing insulin concentration measured in the treatments of 17

Figure 4 .
Figure 4. UPF3 overexpression deteriorates cell viability and reduces insulin content, but not secretion in EndoC-βH3 cells

Figure 5 .
Figure 5. UPF2 knockdown potentiates cytokines suppression of NMD activity and slightly alleviates cytokines toxicity for cell viability and insulin content in EndoC-βH3 cells
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