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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

Reduced poly(A) tail shortening in CNOT3-depleted MEFs stabilizes target mRNAs.(a,b) Control and CNOT3KD MEFs were treated with Act. D. Relative mRNA levels were determined by qPCR at 4 h time intervals after Act. D treatment and normalized to the gapdh mRNA level. mRNA level without Act. D treatment (0 h) was set to 100%. n = 3 for each genotype. All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001 (c) Comparison of poly(A) tail length of mRNAs between control and CNOT3KD MEFs. Total cellular RNA was subjected to PCR-based analysis (see Methods). PCR products without poly(A) regions (−) were also loaded. (d) Lysates from MEFs were immunoprecipitated with control Ig or anti-CNOT3 antibodies. Immunoprecipitates were analyzed by RT-PCR using primers for 3′UTR in the indicated genes.
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f5: Reduced poly(A) tail shortening in CNOT3-depleted MEFs stabilizes target mRNAs.(a,b) Control and CNOT3KD MEFs were treated with Act. D. Relative mRNA levels were determined by qPCR at 4 h time intervals after Act. D treatment and normalized to the gapdh mRNA level. mRNA level without Act. D treatment (0 h) was set to 100%. n = 3 for each genotype. All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001 (c) Comparison of poly(A) tail length of mRNAs between control and CNOT3KD MEFs. Total cellular RNA was subjected to PCR-based analysis (see Methods). PCR products without poly(A) regions (−) were also loaded. (d) Lysates from MEFs were immunoprecipitated with control Ig or anti-CNOT3 antibodies. Immunoprecipitates were analyzed by RT-PCR using primers for 3′UTR in the indicated genes.

Mentions: We analyzed the time course of mRNA expression level following Act. D treatment and found that half-lives of pik3c3, ripk1, ripk3, creb3, dvl2, sirt5 and cdkn1a mRNAs were prolonged in CNOT3-depleted MEFs (Fig. 5a; Supplementary Fig. 5A). In contrast, half-lives of fbxo30, klf9 and zfp292 mRNAs were similar between control and CNOT3-depleted MEFs (Fig. 5b). To verify that stabilization of mRNAs in the absence of CNOT3 is a consequence of poor deadenylation by the CCR4-NOT complex, we analyzed poly(A) tail lengths of pik3c3, ripk1 and ripk3 mRNAs. Their poly(A) tails were significantly longer in CNOT3-depleted MEFs than control MEFs (Fig. 5c). Furthermore, reverse transcription (RT)-PCR analysis of RNAs that immunoprecipitated with anti-CNOT3 antibody showed that CNOT3 physically interacts with pik3c3, ripk1, ripk3, creb3, dvl2, sirt5 and cdkn1a mRNAs (Fig. 5d; Supplementary Fig. 5B). Previous reports showed that the CCR4-NOT complex utilizes the miRNA/Argonaute (Ago) complex and/or an AU-rich element binding protein, such as Zfp36L1, to recognize target mRNAs3536. We found that Zfp36L1 bound to the 3′ UTR of ripk1 and creb3 mRNAs that possess AU-rich elements in vitro (Supplementary Fig. 6A,B). Furthermore, Ago2 bound to the 3′ UTR of pik3c3, ripk1 and creb3mRNA (Supplementary Fig. 6B). These findings suggest that CNOT3 recognizes 3′ UTR sequences of pik3c3, ripk1 and creb3 mRNAs in a manner that is dependent on the miRNA/Ago complex and/or Zfp36L1.


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)

Reduced poly(A) tail shortening in CNOT3-depleted MEFs stabilizes target mRNAs.(a,b) Control and CNOT3KD MEFs were treated with Act. D. Relative mRNA levels were determined by qPCR at 4 h time intervals after Act. D treatment and normalized to the gapdh mRNA level. mRNA level without Act. D treatment (0 h) was set to 100%. n = 3 for each genotype. All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001 (c) Comparison of poly(A) tail length of mRNAs between control and CNOT3KD MEFs. Total cellular RNA was subjected to PCR-based analysis (see Methods). PCR products without poly(A) regions (−) were also loaded. (d) Lysates from MEFs were immunoprecipitated with control Ig or anti-CNOT3 antibodies. Immunoprecipitates were analyzed by RT-PCR using primers for 3′UTR in the indicated genes.
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f5: Reduced poly(A) tail shortening in CNOT3-depleted MEFs stabilizes target mRNAs.(a,b) Control and CNOT3KD MEFs were treated with Act. D. Relative mRNA levels were determined by qPCR at 4 h time intervals after Act. D treatment and normalized to the gapdh mRNA level. mRNA level without Act. D treatment (0 h) was set to 100%. n = 3 for each genotype. All values represent means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001 (c) Comparison of poly(A) tail length of mRNAs between control and CNOT3KD MEFs. Total cellular RNA was subjected to PCR-based analysis (see Methods). PCR products without poly(A) regions (−) were also loaded. (d) Lysates from MEFs were immunoprecipitated with control Ig or anti-CNOT3 antibodies. Immunoprecipitates were analyzed by RT-PCR using primers for 3′UTR in the indicated genes.
Mentions: We analyzed the time course of mRNA expression level following Act. D treatment and found that half-lives of pik3c3, ripk1, ripk3, creb3, dvl2, sirt5 and cdkn1a mRNAs were prolonged in CNOT3-depleted MEFs (Fig. 5a; Supplementary Fig. 5A). In contrast, half-lives of fbxo30, klf9 and zfp292 mRNAs were similar between control and CNOT3-depleted MEFs (Fig. 5b). To verify that stabilization of mRNAs in the absence of CNOT3 is a consequence of poor deadenylation by the CCR4-NOT complex, we analyzed poly(A) tail lengths of pik3c3, ripk1 and ripk3 mRNAs. Their poly(A) tails were significantly longer in CNOT3-depleted MEFs than control MEFs (Fig. 5c). Furthermore, reverse transcription (RT)-PCR analysis of RNAs that immunoprecipitated with anti-CNOT3 antibody showed that CNOT3 physically interacts with pik3c3, ripk1, ripk3, creb3, dvl2, sirt5 and cdkn1a mRNAs (Fig. 5d; Supplementary Fig. 5B). Previous reports showed that the CCR4-NOT complex utilizes the miRNA/Argonaute (Ago) complex and/or an AU-rich element binding protein, such as Zfp36L1, to recognize target mRNAs3536. We found that Zfp36L1 bound to the 3′ UTR of ripk1 and creb3 mRNAs that possess AU-rich elements in vitro (Supplementary Fig. 6A,B). Furthermore, Ago2 bound to the 3′ UTR of pik3c3, ripk1 and creb3mRNA (Supplementary Fig. 6B). These findings suggest that CNOT3 recognizes 3′ UTR sequences of pik3c3, ripk1 and creb3 mRNAs in a manner that is dependent on the miRNA/Ago complex and/or Zfp36L1.

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