Protein & Cell

, Volume 2, Issue 9, pp 755–763 | Cite as

The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

  • Kentaro Ito
  • Akinori Takahashi
  • Masahiro Morita
  • Toru Suzuki
  • Tadashi Yamamoto
Research Article

Abstract

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G1 fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.

Keywords

deadenylation CCR4-NOT small interfering RNA P-bodies apoptosis 

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References

  1. Bartlam, M., and Yamamoto, T. (2010). The structural basis for deadenylation by the CCR4-NOT complex. Protein Cell 1, 443–452.CrossRefGoogle Scholar
  2. Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P., and Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20, 1885–1898.CrossRefGoogle Scholar
  3. Berthet, C., Morera, A.M., Asensio, M.J., Chauvin, M.A., Morel, A.P., Dijoud, F., Magaud, J.P., Durand, P., and Rouault, J.P. (2004). CCR4-associated factor CAF1 is an essential factor for spermatogenesis. Mol Cell Biol 24, 5808–5820.CrossRefGoogle Scholar
  4. Chen, C., Ito, K., Takahashi, A., Suzuki, T., Ge, W., Nakazawa, T., Yamamoto, T., and Yokoyama, K. (2011). Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development. Biophys Biochem Res Commun 411, 360–364.CrossRefGoogle Scholar
  5. Chicoine, J., Benoit, P., Gamberi, C., Paliouras, M., Simonelig, M., and Lasko, P. (2007). Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression. Dev Cell 13, 691–704.CrossRefGoogle Scholar
  6. Collart, M.A. (2003). Global control of gene expression in yeast by the Ccr4-Not complex. Gene 313, 1–16.CrossRefGoogle Scholar
  7. Collart, M.A., and Timmers, H.T. (2004). The eukaryotic Ccr4-not complex: a regulatory platform integrating mRNA metabolism with cellular signaling pathways? Prog Nucleic Acid Res Mol Biol 77, 289–322.CrossRefGoogle Scholar
  8. Eulalio, A., Behm-Ansmant, I., Schweizer, D., and Izaurralde, E. (2007). P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol 27, 3970–3981.CrossRefGoogle Scholar
  9. Garneau, N.L., Wilusz, J., and Wilusz, C.J. (2007). The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 8, 113–126.CrossRefGoogle Scholar
  10. Graces, R., Gillon, W., and Pai, E.F. (2009). Atomic model of human Rcd-1 reveals an armadillo-like-repeat protein with in vitro nucleic acid binding properties. Protein Sci 16, 176–188.CrossRefGoogle Scholar
  11. Harding, H.P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6, 1099–1108.CrossRefGoogle Scholar
  12. Hitomi, J., Katayama, T., Taniguchi, M., Honda, A., Imaizumi, K., and Tohyama, M. (2004). Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci Lett 357, 127–130.CrossRefGoogle Scholar
  13. Ito, K., Inoue, T., Yokoyama, K., Morita, M., Suzuki, T., and Yamamoto, T. (2011). CNOT2 depletion disrupts and inhibits the CCR4-NOT deadenylase complex and induces apoptotic cell death. Genes Cells 16, 368–379.CrossRefGoogle Scholar
  14. Lin, J. H., Li, H., Yasumura, D., Cohen, H. R., Chao, Z., Panning, B., Shokat, K. M., LaVail, M. M., and Walter, P. (2007). IRE1 signaling affects cell fate during the unfolded protein response. Science 318, 944–949.CrossRefGoogle Scholar
  15. Maillet, L., Tu, C., Hong, Y.K., Shuster, E.O., and Collart, M.A. (2000). The essential function of Not1 lies within the Ccr4-Not complex. J Mol Biol 303, 131–143.CrossRefGoogle Scholar
  16. Miyasaka, T., Morita, M., Ito, K., Suzuki, T., Fukuda, H., Takeda, S., Inoue, J., Semba, K., and Yamamoto, T. (2008). Interaction of antiproliferative protein Tob with the CCR4-NOT deadenylase complex. Cancer Sci 99, 755–761.CrossRefGoogle Scholar
  17. Morita, M., Suzuki, T., Nakamura, T., Yokoyama, K., Miyasaka, T., and Yamamoto, T. (2007). Depletion of mammalian CCR4b deadenylase triggers elevation of the p27Kip1 mRNA level and impairs cell growth. Mol Cell Biol 27, 4980–4990.CrossRefGoogle Scholar
  18. Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A., and Yuan, J. (2000). Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403, 98–103.CrossRefGoogle Scholar
  19. Nakamura, T., Yao, R., Ogawa, T., Suzuki, T., Ito, C., Tsunekawa, N., Inoue, K., Ajima, R., Miyasaka, T., Yoshida, Y., et al. (2004). Oligoastheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta. Nat Genet 36, 528–533.CrossRefGoogle Scholar
  20. Neely, G.G., Kuba, K., Cammarato, A., Isobe, K., Amann, S., Zhang, L., Murata, M., Elmén, L., Gupta, V., Arora, S., et al. (2010). A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function. Cell 141, 142–153.CrossRefGoogle Scholar
  21. Sandler, H., Kreth, J., Timmers, H.T.M., and Stoecklin, G. (2011). Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin. Nucleic Acids Res 39, 4373–4386.CrossRefGoogle Scholar
  22. Schröder, M., and Kaufman, R.J. (2005). ER stress and the unfolded protein response. Mutat Res 569, 29–63.CrossRefGoogle Scholar
  23. Sheth, U., and Parker, R. (2003). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808.CrossRefGoogle Scholar
  24. Temme, C., Zaessinger, S., Meyer, S., Simonelig, M., and Wahle, E. (2004). A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J 23, 2862–2871.CrossRefGoogle Scholar
  25. Temme, C., Zhang, L., Kremmer, E., Ihling, C., Chartier, A., Sinz, A., Simonelig, M., and Wahle, E. (2010). Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation. RNA 16, 1356–1370.CrossRefGoogle Scholar
  26. Tucker, M., Staples, R.R., Valencia-Sanchez, M.A., Muhlrad, D., and Parker, R. (2002). Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO J 21, 1427–1436.CrossRefGoogle Scholar
  27. Wang, H., Morita, M., Yang, X., Suzuki, T., Yang, W., Wang, J., Ito, K., Wang, Q., Zhao, C., Bartlam, M., et al. (2010). Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity. EMBO J 29, 2566–2576.CrossRefGoogle Scholar
  28. Winkler, G.S., Mulder, K.W., Bardwell, V.J., Kalkhoven, E., and Timmers, H.T. (2006). Human Ccr4-Not complex is a liganddependent repressor of nuclear receptor-mediated transcription. EMBO J 25, 3089–3099.CrossRefGoogle Scholar
  29. Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R.T., Remotti, H., Stevens, J.L., and Ron, D. (1998). CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev 12, 982–995CrossRefGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Kentaro Ito
    • 1
  • Akinori Takahashi
    • 1
  • Masahiro Morita
    • 1
  • Toru Suzuki
    • 1
  • Tadashi Yamamoto
    • 1
    • 2
  1. 1.Division of Oncology, Department of Cancer Biology, Institute of Medical ScienceUniversity of TokyoTokyoJapan
  2. 2.Cell Signal UnitOkinawa Institute of Science and Technology, 1919-1OkinawaJapan

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