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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 necrosis via the RIPK3-dependent death pathway. (a) Lysates were prepared from MEFs which were infected with retrovirus expressing shRNAs (shControl, shRIPK3-293, shRIPK3- 827 or shRIPK1) and adenovirus (LacZ or Cre) were analyzed by immunoblot. (b) Schematic diagram of positions of photographs. (c) Morphology of CNOT3KD MEFs and control MEFs (Insets) infected with retrovirus-expressing shRNAs which are treated with DMSO or zVAD for 24 h. (d) Cell death assessed by PI uptake via flow cytometry of MEFs transduced with retrovirus (shRNA constructs) and adenovirus (LacZ or Cre) 24 h after an addition of DMSO (−) or zVAD (+). n = 3. All values represent means ± sem. **P < 0.01
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f7: CNOT3-depleted MEFs undergo necrosis via the RIPK3-dependent death pathway. (a) Lysates were prepared from MEFs which were infected with retrovirus expressing shRNAs (shControl, shRIPK3-293, shRIPK3- 827 or shRIPK1) and adenovirus (LacZ or Cre) were analyzed by immunoblot. (b) Schematic diagram of positions of photographs. (c) Morphology of CNOT3KD MEFs and control MEFs (Insets) infected with retrovirus-expressing shRNAs which are treated with DMSO or zVAD for 24 h. (d) Cell death assessed by PI uptake via flow cytometry of MEFs transduced with retrovirus (shRNA constructs) and adenovirus (LacZ or Cre) 24 h after an addition of DMSO (−) or zVAD (+). n = 3. All values represent means ± sem. **P < 0.01

Mentions: The upregulation/stabilization of ripk1, ripk3 and mRNAs for other necrosis-related proteins in CNOT3-depleted MEFs appeared to be the cause of necroptosis (Figs 4d,e and 5a; Supplementary Fig.3). While RIPK1 associates with and activates RIPK3 to promote necroptosis upon stimulation of TNFR1 (ref. 21), RIPK1 inhibits necroptosis through the action of Caspase-8-FLIP complex when RIPK3 is activated independently, such as by TLR ligation or interferon (IFN) stimulation37. Because both mRNAs encoding proteins involved in RIPK1-dependent and RIPK1-independent necroptosis were stabilized in CNOT3-depleted MEFs (Fig. 4d; Supplementary Table 3), we first investigated the effect of expressing shRNA specific for RIPK3 (shRIPK3) on necroptosis in CNOT3-depleted MEFs. We used two different shRNAs that target different sequence segments of RIPK3. Expression of shRIPK3-293 results in strong silencing of RIPK3 (48% reduction), while shRIPK3-827 is less effective in this regard, reducing RIPK3 expression to near to normal levels (16% reduction) in CNOTKD MEFs (Fig. 7a). In the presence of CNOT3, the viability of MEFs in which shRIPK3 was introduced, was similar to that of control shRNA-introduced MEFs (Fig. 7c, insets: compare left top with middle two panels, 7d). Upon CNOT3 depletion, the number of dead cells increased among control shRNA-introduced MEFs, but not in MEFs treated with shRIPK3 (Fig. 7c, compare left top with middle two panels, 7d). Furthermore, many CNOT3-depleted, shRIPK3-treated MEFs were viable in the presence of zVAD, whereas zVAD treatment of CNOT3-depleted MEFs expressing control shRNA resulted in extensive cell death (Fig. 7c, compare right top with middle two panels, 7d). Suppression of RIPK3 significantly, though not completely, restored the proliferation rate of CNOT3-depleted MEFs (Supplementary Fig. 8). The results are similar to the effect of Nec-1 on cell proliferation (Fig. 2d). Importantly, necroptosis is significantly suppressed by both shRIPK3-827 and shRIPK3-293, indicating that reduction of RIPK3 to normal levels in CNOT3KD is sufficient to protect MEFs from necroptosis. These results suggest that CNOT3 suppression-induced stabilization of ripk3 mRNA contributes to necroptosis induction. In contrast, when we suppressed expression of RIPK1 using shRNA specific for RIPK1 (shRIPK1), death of CNOT3-depleted MEFs was promoted rather than inhibited (Fig. 7c, bottom left, 7d). This likely occurred because the anti-necroptotic function of the Caspase-8-FLIP complex could not be promoted by RIPK1 silencing. Similar effects were observed in the presence of zVAD together with shRIPK1 (Fig. 7c, bottom right, 7d).Note that Nec-1 does not inhibit necroptosis in the absence of RIPK1 and Nec-1-bound RIPK1 plays a critical role in necroptosis inhibition37. Consistent with this, necroptosis observed in CNOT3KD MEFs was not suppressed by Nec-1 when RIPK1 was silenced (Supplementary Fig. 9).


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 necrosis via the RIPK3-dependent death pathway. (a) Lysates were prepared from MEFs which were infected with retrovirus expressing shRNAs (shControl, shRIPK3-293, shRIPK3- 827 or shRIPK1) and adenovirus (LacZ or Cre) were analyzed by immunoblot. (b) Schematic diagram of positions of photographs. (c) Morphology of CNOT3KD MEFs and control MEFs (Insets) infected with retrovirus-expressing shRNAs which are treated with DMSO or zVAD for 24 h. (d) Cell death assessed by PI uptake via flow cytometry of MEFs transduced with retrovirus (shRNA constructs) and adenovirus (LacZ or Cre) 24 h after an addition of DMSO (−) or zVAD (+). n = 3. All values represent means ± sem. **P < 0.01
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f7: CNOT3-depleted MEFs undergo necrosis via the RIPK3-dependent death pathway. (a) Lysates were prepared from MEFs which were infected with retrovirus expressing shRNAs (shControl, shRIPK3-293, shRIPK3- 827 or shRIPK1) and adenovirus (LacZ or Cre) were analyzed by immunoblot. (b) Schematic diagram of positions of photographs. (c) Morphology of CNOT3KD MEFs and control MEFs (Insets) infected with retrovirus-expressing shRNAs which are treated with DMSO or zVAD for 24 h. (d) Cell death assessed by PI uptake via flow cytometry of MEFs transduced with retrovirus (shRNA constructs) and adenovirus (LacZ or Cre) 24 h after an addition of DMSO (−) or zVAD (+). n = 3. All values represent means ± sem. **P < 0.01
Mentions: The upregulation/stabilization of ripk1, ripk3 and mRNAs for other necrosis-related proteins in CNOT3-depleted MEFs appeared to be the cause of necroptosis (Figs 4d,e and 5a; Supplementary Fig.3). While RIPK1 associates with and activates RIPK3 to promote necroptosis upon stimulation of TNFR1 (ref. 21), RIPK1 inhibits necroptosis through the action of Caspase-8-FLIP complex when RIPK3 is activated independently, such as by TLR ligation or interferon (IFN) stimulation37. Because both mRNAs encoding proteins involved in RIPK1-dependent and RIPK1-independent necroptosis were stabilized in CNOT3-depleted MEFs (Fig. 4d; Supplementary Table 3), we first investigated the effect of expressing shRNA specific for RIPK3 (shRIPK3) on necroptosis in CNOT3-depleted MEFs. We used two different shRNAs that target different sequence segments of RIPK3. Expression of shRIPK3-293 results in strong silencing of RIPK3 (48% reduction), while shRIPK3-827 is less effective in this regard, reducing RIPK3 expression to near to normal levels (16% reduction) in CNOTKD MEFs (Fig. 7a). In the presence of CNOT3, the viability of MEFs in which shRIPK3 was introduced, was similar to that of control shRNA-introduced MEFs (Fig. 7c, insets: compare left top with middle two panels, 7d). Upon CNOT3 depletion, the number of dead cells increased among control shRNA-introduced MEFs, but not in MEFs treated with shRIPK3 (Fig. 7c, compare left top with middle two panels, 7d). Furthermore, many CNOT3-depleted, shRIPK3-treated MEFs were viable in the presence of zVAD, whereas zVAD treatment of CNOT3-depleted MEFs expressing control shRNA resulted in extensive cell death (Fig. 7c, compare right top with middle two panels, 7d). Suppression of RIPK3 significantly, though not completely, restored the proliferation rate of CNOT3-depleted MEFs (Supplementary Fig. 8). The results are similar to the effect of Nec-1 on cell proliferation (Fig. 2d). Importantly, necroptosis is significantly suppressed by both shRIPK3-827 and shRIPK3-293, indicating that reduction of RIPK3 to normal levels in CNOT3KD is sufficient to protect MEFs from necroptosis. These results suggest that CNOT3 suppression-induced stabilization of ripk3 mRNA contributes to necroptosis induction. In contrast, when we suppressed expression of RIPK1 using shRNA specific for RIPK1 (shRIPK1), death of CNOT3-depleted MEFs was promoted rather than inhibited (Fig. 7c, bottom left, 7d). This likely occurred because the anti-necroptotic function of the Caspase-8-FLIP complex could not be promoted by RIPK1 silencing. Similar effects were observed in the presence of zVAD together with shRIPK1 (Fig. 7c, bottom right, 7d).Note that Nec-1 does not inhibit necroptosis in the absence of RIPK1 and Nec-1-bound RIPK1 plays a critical role in necroptosis inhibition37. Consistent with this, necroptosis observed in CNOT3KD MEFs was not suppressed by Nec-1 when RIPK1 was silenced (Supplementary Fig. 9).

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