Mammalian UPF3A and UPF3B activate NMD independently of their EJC binding

Yi, Zhongxia; Arvola, René M.; Myers, Sean; Cn, Dilsavor; R, Abu Alhasan; Carter, Bayley N; Patton, Robert D.; Bundschuh, Ralf; Singh, Guramrit

doi:10.1101/2021.07.02.450872

Cited by 3 publications

(4 citation statements)

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“…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.…”

Section: Discussionmentioning

confidence: 82%

“…Since we observed in our previous study that cytokine-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) since (11),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 proposed Upf3-dependent and -independent branches of NMD pathway (4, 10, 24). we reasoned that UPF3 regulated NMD activity in β-cells We therefore first measured the UPF3A/B expression level and next investigated the functional impact of overexpressing UPF3A/B on cytokine-mediated suppression of NMD activity in β-cells.…”

Section: Resultsmentioning

confidence: 99%

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

Ghiasi,

Marchetti,

Piemonti

et al. 2023

Preprint

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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 was used to examine the effect of proinflammatory cytokines (Cyt) and/or glucolipotoxicity (GLT) on NMD activity. Gain- or loss-of function of two key NMD components UPF3B and UPF2 was used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing were deployed using standard techniques. Cyt, but not GLT, attenuated NMD activity in insulin-producing cell lines and primary human β-cells. These effects were found to involve ER stress and were associated with downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing, raised or lowered Cyt-induced cell death, respectively, in EndoC-βH3 cells, and were associated with decreased or increased insulin content, respectively. No effects of these manipulations were observed on glucose-stimulated insulin secretion. Transcriptomic analysis revealed that, in contrast to GLT, Cyt increased alternative splicing (AS)- induced exon skipping in the transcript isoforms, and this was potentiated by UPF2 silencing. Gene enrichment analysis identified 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 sensitised β-cells to Cyt cytotoxicity. Cyt 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.

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Section: Discussionmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

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

Ghiasi,

Marchetti,

Piemonti

et al. 2023

Preprint

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“…EndoC-bH3 and INS1(832/13) cells were transfected with siRNAs against SERPINA1 (two species-specific siRNAs for each cell type; si1 and si2) and a nonsilencing siRNA control (NS), incubated for 24 h and exposed to PBS as untreated (Unt) and cytokine combination (Cyt for EndoC-bH3; 3 ng/mL IL-1b + 10 ng/mL IFN-g+ 10 ng/mL TNF-a) (Cyt for INS1(832/13); 150 pg/mL IL-1b + 0.1 ng/mL IFN-g+ 0.1 ng/mL TNF-a) for 72 and 24 h, respectively (see Supplementary Methods). The knockdown efficiency was checked using quantitative WB [Supplementary Figures S8 (i 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 b cells.…”

Section: Discussionmentioning

confidence: 99%

“…Since we observed in our previous study that cytokine-induced ER stress downregulated UPF3B expression in human and rodent b cells, as recovering nitroxidative-driven ER stress using the inducible nitric oxide synthase (iNOS) inhibitor N-methyl-larginine (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 UPF3-independent branches of the NMD pathway (4,10,24). We reasoned that UPF3 regulated NMD activity in b cells.…”

Section: Cytokine-induced Suppression Of Nmd Activity Is Associated W...mentioning

confidence: 99%

Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic β cells

Ghiasi,

Marchetti,

Piemonti

et al. 2024

Front. Endocrinol.

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IntroductionProinflammatory cytokines are implicated in pancreatic ß 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 ß cells.MethodsA luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The 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.ResultsCyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 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 increases 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 the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression.ConclusionOur findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

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UPF3A and UPF3B are redundant and modular activators of nonsense-mediated mRNA decay in human cells

Wallmeroth

Boehm

Lackmann

et al. 2021

Preprint

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The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited normal NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.

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Mammalian UPF3A and UPF3B activate NMD independently of their EJC binding

Cited by 3 publications

References 71 publications

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

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

Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic β cells

UPF3A and UPF3B are redundant and modular activators of nonsense-mediated mRNA decay in human cells

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