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CNOT3 suppression promotes necroptosis by stabilizing mRNAs for cell death-inducing proteins.

Suzuki T, Kikuguchi C, Sharma S, Sasaki T, Tokumasu M, Adachi S, Natsume T, Kanegae Y, Yamamoto T - Sci Rep (2015)

Bottom Line: The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established.The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone.Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death.

View Article: PubMed Central - PubMed

Affiliation: Cell Signal Unit, Okinawa Institute of Science and Technology, 1919-1 Onna-son, Okinawa 904-0495, Japan.

ABSTRACT
The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established. We show here that CNOT3-depleted mouse embryonic fibroblasts (MEFs) undergo cell death. Levels of other complex subunits are decreased in CNOT3-depleted MEFs. The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone. Gene expression profiling reveals that mRNAs encoding cell death-related proteins, including receptor-interacting protein kinase 1 (RIPK1) and RIPK3, are stabilized in CNOT3-depleted MEFs. Some of these mRNAs bind to CNOT3, and in the absence of CNOT3 their poly(A) tails are elongated. Inhibition of RIPK1-RIPK3 signaling by a short-hairpin RNA or a necroptosis inhibitor, necrostatin-1, confers viability upon CNOT3-depleted MEFs. Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death.

No MeSH data available.


Related in: MedlinePlus

Microarray analysis of RNA following transcriptional inhibition identifies CNOT3-regulated mRNAs.(a) Procedure for selection of genes, the stability of which is affected by CNOT3 suppression. (b) Heat map after a hierarchical clustering. A red square indicates a cluster in which transcripts are largely decreased in controls, but not CNOT3KD MEFs after Act. D treatment. (c) Venn diagram illustrating the overlap between the gene selected from (b) (circled red) and genes up-regulated >1.5 fold in CNOT3KD MEFs compared with control MEFs (circled blue). (d) Heat map showing relative expression levels of the overlapped 489 genes in (c). (e) qPCR analysis of mRNAs in control or CNOT3KD MEFs. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. **P < 0.01; ***P < 0.001
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f4: Microarray analysis of RNA following transcriptional inhibition identifies CNOT3-regulated mRNAs.(a) Procedure for selection of genes, the stability of which is affected by CNOT3 suppression. (b) Heat map after a hierarchical clustering. A red square indicates a cluster in which transcripts are largely decreased in controls, but not CNOT3KD MEFs after Act. D treatment. (c) Venn diagram illustrating the overlap between the gene selected from (b) (circled red) and genes up-regulated >1.5 fold in CNOT3KD MEFs compared with control MEFs (circled blue). (d) Heat map showing relative expression levels of the overlapped 489 genes in (c). (e) qPCR analysis of mRNAs in control or CNOT3KD MEFs. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. **P < 0.01; ***P < 0.001

Mentions: Because CNOT3 is involved in regulation of CCR4-NOT deadenylase activity and thereby controls mRNA levels14, we compared gene expression profiles between control and CNOT3-depleted MEFs using Affymetrix microarray technology to elucidate the molecular basis of necroptosis in CNOT3-depleted MEFs. Total RNA samples were prepared from MEFs treated with the transcriptional inhibitor, actinomycin D (Act. D), for 6 and 12 h (Fig. 4a). Hierarchical clustering of gene sets revealed that one cluster of 2,273 transcript probe sets was highly stabilized in CNOT3-depleted MEFs compared with control MEFs (Fig. 4b; Supplementary Table 1). We hypothesized that a significant population, though perhaps not all of these mRNAs, are CNOT3 targets. We then searched for mRNAs that were more highly expressed in CNOT3-depleted MEFs than control MEFs without Act. D treatment. Among the 2,273 probe sets, 489 corresponded to genes that were upregulated more than 1.5-fold in CNOT3-depleted MEFs compared with control MEFs (Fig. 4c; Supplementary Table 2). The upregulated mRNAs encoded proteins involved in regulation of cytoplasm organization, autophagy, cell death, and cell proliferation (Supplementary Table 3). Importantly, necroptosis-related genes, such as ripk1, irak3, tlr2 and tlr3 were included among the upregulated genes (Fig. 4d; Supplementary Table 3). By examining levels of these mRNAs using quantitative real-time polymerase chain reaction (qPCR), we found that creb3, pik3c3, ripk1, dvl2, sirt5 and cdkn1a mRNA levels were significantly elevated in CNOT3-depleted MEFs (Fig. 4e; Supplementary Fig. 4A). We anticipated that the upregulated mRNAs might also include an important necroptosis-related gene, ripk3 mRNA. While ripk3 mRNA was recognized by the microarray analysis, it was present at such low levels in WT MEFs that it was impossible to determine whether there had been a change in expression level. Therefore, we directly measured ripk3 mRNA by qPCR to ascertain its higher expression level in CNOT3-depleted MEFs (Supplementary Fig. 4A). Consistent with this, both RIPK1 and RIPK3 proteins increased following CNOT3 depletion (Supplementary Fig. 4B). Note that transcripts, such as fbxo30, klf9 or zfp292, that have short half-lives both in the presence and absence of CNOT3 in microarray results, were almost the same in control and CNOT3-depleted MEFs (Fig. 4e).


CNOT3 suppression promotes necroptosis by stabilizing mRNAs for cell death-inducing proteins.

Suzuki T, Kikuguchi C, Sharma S, Sasaki T, Tokumasu M, Adachi S, Natsume T, Kanegae Y, Yamamoto T - Sci Rep (2015)

Microarray analysis of RNA following transcriptional inhibition identifies CNOT3-regulated mRNAs.(a) Procedure for selection of genes, the stability of which is affected by CNOT3 suppression. (b) Heat map after a hierarchical clustering. A red square indicates a cluster in which transcripts are largely decreased in controls, but not CNOT3KD MEFs after Act. D treatment. (c) Venn diagram illustrating the overlap between the gene selected from (b) (circled red) and genes up-regulated >1.5 fold in CNOT3KD MEFs compared with control MEFs (circled blue). (d) Heat map showing relative expression levels of the overlapped 489 genes in (c). (e) qPCR analysis of mRNAs in control or CNOT3KD MEFs. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. **P < 0.01; ***P < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594005&req=5

f4: Microarray analysis of RNA following transcriptional inhibition identifies CNOT3-regulated mRNAs.(a) Procedure for selection of genes, the stability of which is affected by CNOT3 suppression. (b) Heat map after a hierarchical clustering. A red square indicates a cluster in which transcripts are largely decreased in controls, but not CNOT3KD MEFs after Act. D treatment. (c) Venn diagram illustrating the overlap between the gene selected from (b) (circled red) and genes up-regulated >1.5 fold in CNOT3KD MEFs compared with control MEFs (circled blue). (d) Heat map showing relative expression levels of the overlapped 489 genes in (c). (e) qPCR analysis of mRNAs in control or CNOT3KD MEFs. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. **P < 0.01; ***P < 0.001
Mentions: Because CNOT3 is involved in regulation of CCR4-NOT deadenylase activity and thereby controls mRNA levels14, we compared gene expression profiles between control and CNOT3-depleted MEFs using Affymetrix microarray technology to elucidate the molecular basis of necroptosis in CNOT3-depleted MEFs. Total RNA samples were prepared from MEFs treated with the transcriptional inhibitor, actinomycin D (Act. D), for 6 and 12 h (Fig. 4a). Hierarchical clustering of gene sets revealed that one cluster of 2,273 transcript probe sets was highly stabilized in CNOT3-depleted MEFs compared with control MEFs (Fig. 4b; Supplementary Table 1). We hypothesized that a significant population, though perhaps not all of these mRNAs, are CNOT3 targets. We then searched for mRNAs that were more highly expressed in CNOT3-depleted MEFs than control MEFs without Act. D treatment. Among the 2,273 probe sets, 489 corresponded to genes that were upregulated more than 1.5-fold in CNOT3-depleted MEFs compared with control MEFs (Fig. 4c; Supplementary Table 2). The upregulated mRNAs encoded proteins involved in regulation of cytoplasm organization, autophagy, cell death, and cell proliferation (Supplementary Table 3). Importantly, necroptosis-related genes, such as ripk1, irak3, tlr2 and tlr3 were included among the upregulated genes (Fig. 4d; Supplementary Table 3). By examining levels of these mRNAs using quantitative real-time polymerase chain reaction (qPCR), we found that creb3, pik3c3, ripk1, dvl2, sirt5 and cdkn1a mRNA levels were significantly elevated in CNOT3-depleted MEFs (Fig. 4e; Supplementary Fig. 4A). We anticipated that the upregulated mRNAs might also include an important necroptosis-related gene, ripk3 mRNA. While ripk3 mRNA was recognized by the microarray analysis, it was present at such low levels in WT MEFs that it was impossible to determine whether there had been a change in expression level. Therefore, we directly measured ripk3 mRNA by qPCR to ascertain its higher expression level in CNOT3-depleted MEFs (Supplementary Fig. 4A). Consistent with this, both RIPK1 and RIPK3 proteins increased following CNOT3 depletion (Supplementary Fig. 4B). Note that transcripts, such as fbxo30, klf9 or zfp292, that have short half-lives both in the presence and absence of CNOT3 in microarray results, were almost the same in control and CNOT3-depleted MEFs (Fig. 4e).

Bottom Line: The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established.The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone.Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death.

View Article: PubMed Central - PubMed

Affiliation: Cell Signal Unit, Okinawa Institute of Science and Technology, 1919-1 Onna-son, Okinawa 904-0495, Japan.

ABSTRACT
The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established. We show here that CNOT3-depleted mouse embryonic fibroblasts (MEFs) undergo cell death. Levels of other complex subunits are decreased in CNOT3-depleted MEFs. The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone. Gene expression profiling reveals that mRNAs encoding cell death-related proteins, including receptor-interacting protein kinase 1 (RIPK1) and RIPK3, are stabilized in CNOT3-depleted MEFs. Some of these mRNAs bind to CNOT3, and in the absence of CNOT3 their poly(A) tails are elongated. Inhibition of RIPK1-RIPK3 signaling by a short-hairpin RNA or a necroptosis inhibitor, necrostatin-1, confers viability upon CNOT3-depleted MEFs. Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death.

No MeSH data available.


Related in: MedlinePlus