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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

Both an N-terminal and C-terminal region are required for CNOT3-dependent mRNA decay.Decay curves of CNOT3 targets in CNOT3loxP/loxP MEFs transduced with retroviruses (mock or CNOT3 constructs) and adenoviruses (Ad-LacZ or Ad-Cre) determined as in Fig. 4a. n = 3. WT CNOT3 expression in CNOT3KD MEFs (red lines in left middle graphs), but not CNOTdC and CNOTdN expression (red lines in right middle and rightmost graphs) restored MEF half-lives to control levels (blue lines in leftmost graph. CNOT3dN expression in control MEFs stabilized mRNAs (compare blue lines in between leftmost and rightmost graphs). All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001
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f6: Both an N-terminal and C-terminal region are required for CNOT3-dependent mRNA decay.Decay curves of CNOT3 targets in CNOT3loxP/loxP MEFs transduced with retroviruses (mock or CNOT3 constructs) and adenoviruses (Ad-LacZ or Ad-Cre) determined as in Fig. 4a. n = 3. WT CNOT3 expression in CNOT3KD MEFs (red lines in left middle graphs), but not CNOTdC and CNOTdN expression (red lines in right middle and rightmost graphs) restored MEF half-lives to control levels (blue lines in leftmost graph. CNOT3dN expression in control MEFs stabilized mRNAs (compare blue lines in between leftmost and rightmost graphs). All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001

Mentions: Next, we reintroduced WT CNOT3 into CNOT3-depleted MEFs. The elongated half-lives of creb3, pik3c3 and ripk1 mRNAs were shortened and were comparable to those of control MEFs (Fig. 6, left middle). In contrast, expression of CNOT3dC or CNOT3dN did not restore the half-lives of these mRNAs to control levels (Fig. 6, right middle and rightmost). Moreover, expression of CNOT3dN in control MEFs resulted in a significant stabilization of transcripts (Fig. 6, compare blue lines between Mock and CNOT3dN expressing MEFs). We noted that overexpression of WT CNOT3 transgene in MEFs resulted in stabilization of target mRNAs, albeit slightly, compared to mock retrovirus-infected MEFs (Fig. 6). By performing gel filtration analysis of lysates from control and WT-CNOT3-transduced cells, we found that overexpressed WT CNOT3 formed a smaller complex (~600 kDa) in addition to intact CCR4-NOT complex (Supplementary Fig. 7A). The smaller complex was purified by immunoprecipitation using anti-Flag antibody and the proteins in the complex were subjected to SDS polyacrylamide gel electrophoresis. Silver staining of the gel revealed that CNOT3 was predominantly present, suggesting that the ~600 kDa complex represent a CNOT3 oligomers (Supplementary Fig. 7B). These data suggested that the smaller complex is a CNOT3 oligomer that would interfere with function of the CCR4-NOT complex. Collectively, the effect of CNOT3 (WT and mutants) on cell viability is clearly correlated with its ability to degrade target mRNA levels.


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)

Both an N-terminal and C-terminal region are required for CNOT3-dependent mRNA decay.Decay curves of CNOT3 targets in CNOT3loxP/loxP MEFs transduced with retroviruses (mock or CNOT3 constructs) and adenoviruses (Ad-LacZ or Ad-Cre) determined as in Fig. 4a. n = 3. WT CNOT3 expression in CNOT3KD MEFs (red lines in left middle graphs), but not CNOTdC and CNOTdN expression (red lines in right middle and rightmost graphs) restored MEF half-lives to control levels (blue lines in leftmost graph. CNOT3dN expression in control MEFs stabilized mRNAs (compare blue lines in between leftmost and rightmost graphs). All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Both an N-terminal and C-terminal region are required for CNOT3-dependent mRNA decay.Decay curves of CNOT3 targets in CNOT3loxP/loxP MEFs transduced with retroviruses (mock or CNOT3 constructs) and adenoviruses (Ad-LacZ or Ad-Cre) determined as in Fig. 4a. n = 3. WT CNOT3 expression in CNOT3KD MEFs (red lines in left middle graphs), but not CNOTdC and CNOTdN expression (red lines in right middle and rightmost graphs) restored MEF half-lives to control levels (blue lines in leftmost graph. CNOT3dN expression in control MEFs stabilized mRNAs (compare blue lines in between leftmost and rightmost graphs). All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001
Mentions: Next, we reintroduced WT CNOT3 into CNOT3-depleted MEFs. The elongated half-lives of creb3, pik3c3 and ripk1 mRNAs were shortened and were comparable to those of control MEFs (Fig. 6, left middle). In contrast, expression of CNOT3dC or CNOT3dN did not restore the half-lives of these mRNAs to control levels (Fig. 6, right middle and rightmost). Moreover, expression of CNOT3dN in control MEFs resulted in a significant stabilization of transcripts (Fig. 6, compare blue lines between Mock and CNOT3dN expressing MEFs). We noted that overexpression of WT CNOT3 transgene in MEFs resulted in stabilization of target mRNAs, albeit slightly, compared to mock retrovirus-infected MEFs (Fig. 6). By performing gel filtration analysis of lysates from control and WT-CNOT3-transduced cells, we found that overexpressed WT CNOT3 formed a smaller complex (~600 kDa) in addition to intact CCR4-NOT complex (Supplementary Fig. 7A). The smaller complex was purified by immunoprecipitation using anti-Flag antibody and the proteins in the complex were subjected to SDS polyacrylamide gel electrophoresis. Silver staining of the gel revealed that CNOT3 was predominantly present, suggesting that the ~600 kDa complex represent a CNOT3 oligomers (Supplementary Fig. 7B). These data suggested that the smaller complex is a CNOT3 oligomer that would interfere with function of the CCR4-NOT complex. Collectively, the effect of CNOT3 (WT and mutants) on cell viability is clearly correlated with its ability to degrade target mRNA levels.

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