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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

CNOT3 deficiency decreases other CCR4-NOT complex subunits and cell viability.(a) WT (+/+) and CNOT3loxP/loxP MEFs were infected with mock- or Cre-expressing retrovirus. Cell lysates were analyzed by immunoblot. (b) Cell morphology 4 days after infection. Photographs are at the same magnification. Dead cells that were about to lose adhesion were observed in CNOT3loxP/loxP:Cre MEFs. (c) CNOT3loxP/loxP MEFs infected with mock- (Control) or Cre-expressing retrovirus (CNOT3KD) were lysed 4 days after infection, and lysates were analyzed by immunoblot. (d) qPCR analysis in control or CNOT3KD MEFs 4 days after infection. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. *P < 0.05; ***P < 0.001
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f1: CNOT3 deficiency decreases other CCR4-NOT complex subunits and cell viability.(a) WT (+/+) and CNOT3loxP/loxP MEFs were infected with mock- or Cre-expressing retrovirus. Cell lysates were analyzed by immunoblot. (b) Cell morphology 4 days after infection. Photographs are at the same magnification. Dead cells that were about to lose adhesion were observed in CNOT3loxP/loxP:Cre MEFs. (c) CNOT3loxP/loxP MEFs infected with mock- (Control) or Cre-expressing retrovirus (CNOT3KD) were lysed 4 days after infection, and lysates were analyzed by immunoblot. (d) qPCR analysis in control or CNOT3KD MEFs 4 days after infection. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. *P < 0.05; ***P < 0.001

Mentions: cnot3-deficient mice die in embryo14, which prevented us from studying molecular physiological roles of CNOT3 in tissues and cells. To circumvent this obstacle, we used mice carrying a floxed allele of CNOT3 (CNOT3loxP/loxP)14. Primary embryonic fibroblasts were prepared from mice and were then infected with a retrovirus expressing the Cre recombinase to produce CNOT3-depleted MEFs. Immunoblot analysis revealed that CNOT3-depletion was successful (Fig. 1a). Upon CNOT3 depletion, MEFs underwent cell death, as indicated by the appearance of floating and aggregating cells (Fig. 1b). These outcomes were not observed in WT MEFs infected with mock or Cre-expressing retrovirus, or in CNOT3loxP/loxP MEFs infected with mock retrovirus (Fig. 1b). Hereafter, mock virus-infected CNOT3loxP/loxP MEFs are referred to as control MEFs.


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)

CNOT3 deficiency decreases other CCR4-NOT complex subunits and cell viability.(a) WT (+/+) and CNOT3loxP/loxP MEFs were infected with mock- or Cre-expressing retrovirus. Cell lysates were analyzed by immunoblot. (b) Cell morphology 4 days after infection. Photographs are at the same magnification. Dead cells that were about to lose adhesion were observed in CNOT3loxP/loxP:Cre MEFs. (c) CNOT3loxP/loxP MEFs infected with mock- (Control) or Cre-expressing retrovirus (CNOT3KD) were lysed 4 days after infection, and lysates were analyzed by immunoblot. (d) qPCR analysis in control or CNOT3KD MEFs 4 days after infection. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. *P < 0.05; ***P < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: CNOT3 deficiency decreases other CCR4-NOT complex subunits and cell viability.(a) WT (+/+) and CNOT3loxP/loxP MEFs were infected with mock- or Cre-expressing retrovirus. Cell lysates were analyzed by immunoblot. (b) Cell morphology 4 days after infection. Photographs are at the same magnification. Dead cells that were about to lose adhesion were observed in CNOT3loxP/loxP:Cre MEFs. (c) CNOT3loxP/loxP MEFs infected with mock- (Control) or Cre-expressing retrovirus (CNOT3KD) were lysed 4 days after infection, and lysates were analyzed by immunoblot. (d) qPCR analysis in control or CNOT3KD MEFs 4 days after infection. gapdh mRNA levels were used for normalization. n = 3 for each genotype. All values represent means + sem. *P < 0.05; ***P < 0.001
Mentions: cnot3-deficient mice die in embryo14, which prevented us from studying molecular physiological roles of CNOT3 in tissues and cells. To circumvent this obstacle, we used mice carrying a floxed allele of CNOT3 (CNOT3loxP/loxP)14. Primary embryonic fibroblasts were prepared from mice and were then infected with a retrovirus expressing the Cre recombinase to produce CNOT3-depleted MEFs. Immunoblot analysis revealed that CNOT3-depletion was successful (Fig. 1a). Upon CNOT3 depletion, MEFs underwent cell death, as indicated by the appearance of floating and aggregating cells (Fig. 1b). These outcomes were not observed in WT MEFs infected with mock or Cre-expressing retrovirus, or in CNOT3loxP/loxP MEFs infected with mock retrovirus (Fig. 1b). Hereafter, mock virus-infected CNOT3loxP/loxP MEFs are referred to as control MEFs.

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