Dataset Information

MRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.

ABSTRACT: mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon-exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5'-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.

SUBMITTER: Jakobson M

PROVIDER: S-EPMC3288346 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.

Jakobson M M Lintulahti A A Arumäe U U

Cell death & disease 20120202

mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon-exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer r ...[more]

PMID: 22297299

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Project description: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.

Dataset Information

MRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.

Publications

mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.

Similar Datasets

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