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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

Activity of the CCR4-NOT complex depends upon a complete CNOT3 C-terminus and CNOT3 function requires an intact N-terminus.(a) Schematic diagram of CNOT3. CNOT3 consists of an N-terminal coiled-coil domain, a linker region and a Not box region that shares homology with CNOT2. Numbers above the protein outline represent amino acid positions at domain boundaries. (b) Cell death assessed by PI uptake using flow cytometry of CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). n = 3. (c) Cell morphology 4 days after adenovirus infection. (d) Growth curves corresponding to CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). Day 0 corresponds to 2 days after adenovirus infection. Each time point was determined in triplicate. Growth curves of control cells, Mock/LacZ (blue line) and Mock/Cre (red line), are blotted to every graph for comparison. All values represent means ± sem.*P < 0.05; **P < 0.01. (e) Lysates were prepared from cells infected with retroviruses (CNOT3 constructs) and adenoviruses (LacZ or Cre) and analyzed by immunoblot. The arrow indicates a nonspecific signal. (f) Lysates prepared in (e) were immunoprecipitated with anti-FLAG antibody and immunoprecipitates were analyzed by immunoblot. *IgG heavy and light chains.
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f3: Activity of the CCR4-NOT complex depends upon a complete CNOT3 C-terminus and CNOT3 function requires an intact N-terminus.(a) Schematic diagram of CNOT3. CNOT3 consists of an N-terminal coiled-coil domain, a linker region and a Not box region that shares homology with CNOT2. Numbers above the protein outline represent amino acid positions at domain boundaries. (b) Cell death assessed by PI uptake using flow cytometry of CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). n = 3. (c) Cell morphology 4 days after adenovirus infection. (d) Growth curves corresponding to CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). Day 0 corresponds to 2 days after adenovirus infection. Each time point was determined in triplicate. Growth curves of control cells, Mock/LacZ (blue line) and Mock/Cre (red line), are blotted to every graph for comparison. All values represent means ± sem.*P < 0.05; **P < 0.01. (e) Lysates were prepared from cells infected with retroviruses (CNOT3 constructs) and adenoviruses (LacZ or Cre) and analyzed by immunoblot. The arrow indicates a nonspecific signal. (f) Lysates prepared in (e) were immunoprecipitated with anti-FLAG antibody and immunoprecipitates were analyzed by immunoblot. *IgG heavy and light chains.

Mentions: The C-terminal region of CNOT3, known as the Not box, is required for its interaction with both CNOT1 and CNOT2 (ref. 33). We investigated whether the Not box is necessary for cell viability and for maintaining complex integrity. To do so, we introduced either FLAG epitope-tagged WT CNOT3 or a CNOT3 mutant lacking the 145 C-terminal amino acids (the Not box) (mutant dC, Fig. 3a) into CNOT3-depleted MEFs using a recombinant retrovirus. In this set of experiments, we used a recombinant adenovirus expressing Cre to delete the cnot3 gene34. When WT CNOT3 was reintroduced into CNOT3-depleted MEFs, cell viability was comparable to that of control MEFs (Fig. 3b,c). Reintroduction of WT CNOT3 also rescued the growth capacity of CNOT3-depleted cells (Fig. 3d, left graph). In contrast, expression of CNOT3dC did not rescue these defects (Fig. 3b–d). We also found that introduction of WT CNOT3, but not CNOT3dC, in CNOT3-depleted MEFs rescued expression of all CCR4-NOT complex subunits (Fig. 3e, left panels). Importantly, immunoblot analysis of anti-FLAG immunoprecipitates from CNOT3-depleted cell lysates revealed that CNOT3 was associated with all other subunits when WT CNOT3 was reintroduced, suggesting the formation of complete CCR4-NOT complexes (Fig. 3f, left panels). In contrast, complex formation did not appear to occur in CNOT3dC-introduced cells (Fig. 3f, left panels). These data suggest that the C-terminal region of CNOT3 is necessary for formation of a complete CCR4-NOT complex and for maintaining the expression of other subunits. In summary, the C-terminal region of CNOT3 is indispensable for CCR4-NOT complex integrity and cell viability.


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)

Activity of the CCR4-NOT complex depends upon a complete CNOT3 C-terminus and CNOT3 function requires an intact N-terminus.(a) Schematic diagram of CNOT3. CNOT3 consists of an N-terminal coiled-coil domain, a linker region and a Not box region that shares homology with CNOT2. Numbers above the protein outline represent amino acid positions at domain boundaries. (b) Cell death assessed by PI uptake using flow cytometry of CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). n = 3. (c) Cell morphology 4 days after adenovirus infection. (d) Growth curves corresponding to CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). Day 0 corresponds to 2 days after adenovirus infection. Each time point was determined in triplicate. Growth curves of control cells, Mock/LacZ (blue line) and Mock/Cre (red line), are blotted to every graph for comparison. All values represent means ± sem.*P < 0.05; **P < 0.01. (e) Lysates were prepared from cells infected with retroviruses (CNOT3 constructs) and adenoviruses (LacZ or Cre) and analyzed by immunoblot. The arrow indicates a nonspecific signal. (f) Lysates prepared in (e) were immunoprecipitated with anti-FLAG antibody and immunoprecipitates were analyzed by immunoblot. *IgG heavy and light chains.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4594005&req=5

f3: Activity of the CCR4-NOT complex depends upon a complete CNOT3 C-terminus and CNOT3 function requires an intact N-terminus.(a) Schematic diagram of CNOT3. CNOT3 consists of an N-terminal coiled-coil domain, a linker region and a Not box region that shares homology with CNOT2. Numbers above the protein outline represent amino acid positions at domain boundaries. (b) Cell death assessed by PI uptake using flow cytometry of CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). n = 3. (c) Cell morphology 4 days after adenovirus infection. (d) Growth curves corresponding to CNOT3loxP/loxP MEFs transduced with retroviruses (CNOT3 constructs)/adenoviruses (LacZ or Cre). Day 0 corresponds to 2 days after adenovirus infection. Each time point was determined in triplicate. Growth curves of control cells, Mock/LacZ (blue line) and Mock/Cre (red line), are blotted to every graph for comparison. All values represent means ± sem.*P < 0.05; **P < 0.01. (e) Lysates were prepared from cells infected with retroviruses (CNOT3 constructs) and adenoviruses (LacZ or Cre) and analyzed by immunoblot. The arrow indicates a nonspecific signal. (f) Lysates prepared in (e) were immunoprecipitated with anti-FLAG antibody and immunoprecipitates were analyzed by immunoblot. *IgG heavy and light chains.
Mentions: The C-terminal region of CNOT3, known as the Not box, is required for its interaction with both CNOT1 and CNOT2 (ref. 33). We investigated whether the Not box is necessary for cell viability and for maintaining complex integrity. To do so, we introduced either FLAG epitope-tagged WT CNOT3 or a CNOT3 mutant lacking the 145 C-terminal amino acids (the Not box) (mutant dC, Fig. 3a) into CNOT3-depleted MEFs using a recombinant retrovirus. In this set of experiments, we used a recombinant adenovirus expressing Cre to delete the cnot3 gene34. When WT CNOT3 was reintroduced into CNOT3-depleted MEFs, cell viability was comparable to that of control MEFs (Fig. 3b,c). Reintroduction of WT CNOT3 also rescued the growth capacity of CNOT3-depleted cells (Fig. 3d, left graph). In contrast, expression of CNOT3dC did not rescue these defects (Fig. 3b–d). We also found that introduction of WT CNOT3, but not CNOT3dC, in CNOT3-depleted MEFs rescued expression of all CCR4-NOT complex subunits (Fig. 3e, left panels). Importantly, immunoblot analysis of anti-FLAG immunoprecipitates from CNOT3-depleted cell lysates revealed that CNOT3 was associated with all other subunits when WT CNOT3 was reintroduced, suggesting the formation of complete CCR4-NOT complexes (Fig. 3f, left panels). In contrast, complex formation did not appear to occur in CNOT3dC-introduced cells (Fig. 3f, left panels). These data suggest that the C-terminal region of CNOT3 is necessary for formation of a complete CCR4-NOT complex and for maintaining the expression of other subunits. In summary, the C-terminal region of CNOT3 is indispensable for CCR4-NOT complex integrity and cell viability.

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