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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-depleted MEFs undergo necroptosis.(a) Morphology of control and CNOT3-depleted MEFs (CNOT3KD) 2 days after treatment with dimethyl sulfoxide (DMSO), zVAD, Nec-1, or a combination thereof. Photographs are at the same magnification. (b) Cell death assessed by propidium iodide (PI) uptake using flow cytometry of treated MEFs at the indicated time points after an addition of the indicated chemicals. n = 3. (c) Transmission electron microscopy of control (left panels) and CNOT3KD MEFs (right panels). (d) Growth curves corresponding to control (left) and CNOT3KD MEFs (right) treated with indicated chemicals. Day 0 corresponds to 4 days after retrovirus infection. Each time point was determined in triplicate. All values represent means ± sem. **P < 0.01; ***P < 0.001
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f2: CNOT3-depleted MEFs undergo necroptosis.(a) Morphology of control and CNOT3-depleted MEFs (CNOT3KD) 2 days after treatment with dimethyl sulfoxide (DMSO), zVAD, Nec-1, or a combination thereof. Photographs are at the same magnification. (b) Cell death assessed by propidium iodide (PI) uptake using flow cytometry of treated MEFs at the indicated time points after an addition of the indicated chemicals. n = 3. (c) Transmission electron microscopy of control (left panels) and CNOT3KD MEFs (right panels). (d) Growth curves corresponding to control (left) and CNOT3KD MEFs (right) treated with indicated chemicals. Day 0 corresponds to 4 days after retrovirus infection. Each time point was determined in triplicate. All values represent means ± sem. **P < 0.01; ***P < 0.001

Mentions: Because apoptosis is induced by suppression of CNOT1 or CNOT2 in HeLa cells711, we speculated that depletion of CNOT3 might have a similar effect in MEFs. To investigate whether cell death observed in CNOT3-depleted MEFs is caused by apoptosis, we treated cells with zVAD. While zVAD did not affect the viability of control MEFs, it increased cell death induced by CNOT3 depletion (Fig. 2a,b). zVAD can trigger or enhance necrotic cell death by suppressing the Caspase-8-mediated anti-necrotic pathway23293031. We then treated cells with Necrostatin-1 (Nec-1), which inhibits necroptosis32. Viability of CNOT3-depleted MEFs improved in the presence of Nec-1 (Fig. 2a,b, compare DMSO- and Nec-1-treated CNOT3KD). Additionally, Nec-1 efficiently inhibited zVAD-promoted death of CNOT3-depleted MEFs (Fig. 2a,b, compare zVAD- and zVAD+Nec1-treated CNOT3KD). These results indicate that CNOT3-depleted MEFs underwent necroptosis. Consistent with this finding, electron microscopy revealed that CNOT3-depleted MEFs manifested several necrotic features, such as loss of organelles and subcellular structures and disruption of the plasma membrane (Fig. 2c). Furthermore, neither caspase-8 nor caspase-3, which participate in apoptosis, was significantly activated in CNOT3-depleted MEFs (Supplementary Fig. 2). On day 3 after zVAD treatment that suppresses the anti-necroptotic pathway, surviving cell populations increased (Fig. 2b). We speculate that those cells may have escaped Cre-mediated deletion of the cnot3 gene. Indeed, the surviving cells expressed CNOT3 at levels comparable to those in control MEFs (Supplementary Fig.3).


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-depleted MEFs undergo necroptosis.(a) Morphology of control and CNOT3-depleted MEFs (CNOT3KD) 2 days after treatment with dimethyl sulfoxide (DMSO), zVAD, Nec-1, or a combination thereof. Photographs are at the same magnification. (b) Cell death assessed by propidium iodide (PI) uptake using flow cytometry of treated MEFs at the indicated time points after an addition of the indicated chemicals. n = 3. (c) Transmission electron microscopy of control (left panels) and CNOT3KD MEFs (right panels). (d) Growth curves corresponding to control (left) and CNOT3KD MEFs (right) treated with indicated chemicals. Day 0 corresponds to 4 days after retrovirus infection. Each time point was determined in triplicate. All values represent means ± sem. **P < 0.01; ***P < 0.001
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f2: CNOT3-depleted MEFs undergo necroptosis.(a) Morphology of control and CNOT3-depleted MEFs (CNOT3KD) 2 days after treatment with dimethyl sulfoxide (DMSO), zVAD, Nec-1, or a combination thereof. Photographs are at the same magnification. (b) Cell death assessed by propidium iodide (PI) uptake using flow cytometry of treated MEFs at the indicated time points after an addition of the indicated chemicals. n = 3. (c) Transmission electron microscopy of control (left panels) and CNOT3KD MEFs (right panels). (d) Growth curves corresponding to control (left) and CNOT3KD MEFs (right) treated with indicated chemicals. Day 0 corresponds to 4 days after retrovirus infection. Each time point was determined in triplicate. All values represent means ± sem. **P < 0.01; ***P < 0.001
Mentions: Because apoptosis is induced by suppression of CNOT1 or CNOT2 in HeLa cells711, we speculated that depletion of CNOT3 might have a similar effect in MEFs. To investigate whether cell death observed in CNOT3-depleted MEFs is caused by apoptosis, we treated cells with zVAD. While zVAD did not affect the viability of control MEFs, it increased cell death induced by CNOT3 depletion (Fig. 2a,b). zVAD can trigger or enhance necrotic cell death by suppressing the Caspase-8-mediated anti-necrotic pathway23293031. We then treated cells with Necrostatin-1 (Nec-1), which inhibits necroptosis32. Viability of CNOT3-depleted MEFs improved in the presence of Nec-1 (Fig. 2a,b, compare DMSO- and Nec-1-treated CNOT3KD). Additionally, Nec-1 efficiently inhibited zVAD-promoted death of CNOT3-depleted MEFs (Fig. 2a,b, compare zVAD- and zVAD+Nec1-treated CNOT3KD). These results indicate that CNOT3-depleted MEFs underwent necroptosis. Consistent with this finding, electron microscopy revealed that CNOT3-depleted MEFs manifested several necrotic features, such as loss of organelles and subcellular structures and disruption of the plasma membrane (Fig. 2c). Furthermore, neither caspase-8 nor caspase-3, which participate in apoptosis, was significantly activated in CNOT3-depleted MEFs (Supplementary Fig. 2). On day 3 after zVAD treatment that suppresses the anti-necroptotic pathway, surviving cell populations increased (Fig. 2b). We speculate that those cells may have escaped Cre-mediated deletion of the cnot3 gene. Indeed, the surviving cells expressed CNOT3 at levels comparable to those in control MEFs (Supplementary Fig.3).

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