Volume 9, Issue 2 (Vol.9 No.2 Jul 2020)                   rbmb.net 2020, 9(2): 163-170 | Back to browse issues page

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Eskandarian S, Grand R, Irani S, Saeedi M, Mirfakhraie R. Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR). rbmb.net. 2020; 9 (2) :163-170
URL: http://rbmb.net/article-1-488-en.html
Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
Abstract:   (1026 Views)
Background: The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR).

Methods: Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days’ post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8-depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control.

Results: Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of gH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells.

Conclusions: We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.
Full-Text [PDF 244 kb]   (351 Downloads)    
Type of Article: Original Article | Subject: Molecular Biology
Received: 2020/04/17 | Accepted: 2020/05/3 | Published: 2020/10/7

1. Collart MA, Panasenko OO. The Ccr4-Not complex. Gene. 2012;492(1):42-53. [DOI:10.1016/j.gene.2011.09.033] [PMID]
2. Collart MA. The Ccr4-Not complex is a key regulator of eukaryotic gene expression. Wiley Interdiscip Rev RNA. 2016;7(4):438-454. [DOI:10.1002/wrna.1332] [PMID] [PMCID]
3. Miller JE, Reese JC. Ccr4-Not complex: the control freak of eukaryotic cells. Crit Rev Biochem Mol Biol. 2012;47(4):315-33. [DOI:10.3109/10409238.2012.667214] [PMID] [PMCID]
4. Lau NC, Kolkman A, van Schaik FM, Mulder KW, Pijnappel WP, Heck AJ, Timmers HT. Human Ccr4-Not complexes contain variable deadenylase subunits. Biochem J. 2009; 422(3):443-53. [DOI:10.1042/BJ20090500] [PMID]
5. Boland A, Chen Y, Raisch T, Jonas S, Kuzuoğlu-Öztürk D, Wohlbold L, et al. Structure and assembly of the NOT module of the human CCR4-NOT complex. Nat Struct Mol Biol. 2013;20(11):1289-97. [DOI:10.1038/nsmb.2681] [PMID]
6. Temme C, Zaessinger S, Meyer S, Simonelig M, Wahle E. A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J. 2004;23(14):2862-2871. [DOI:10.1038/sj.emboj.7600273] [PMID] [PMCID]
7. Ito K, Takahashi A, Morita M, Suzuki T, Yamamoto. The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability. Protein Cell. 2011;2(9):755-763. [DOI:10.1007/s13238-011-1092-4] [PMID] [PMCID]
8. Panasenko OO. The role of the E3 ligase Not4 in cotranslational quality control. Front genet.2014;19(5):141. [DOI:10.3389/fgene.2014.00141] [PMID] [PMCID]
9. Maryati M, Airhihen B, Winkler GS. The enzyme activities of Caf1 and Ccr4 are both required for deadenylation by the human Ccr4-Not nuclease module. Biochem J. 2015;469(pt 1):169-176. [DOI:10.1042/BJ20150304] [PMID] [PMCID]
10. Mark Bartlam TY. The structural basis for deadenylation by the CCR4-NOT complex. Protein Cell. 2010;1(5):443-452. [DOI:10.1007/s13238-010-0060-8] [PMID] [PMCID]
11. Aslam A, Mittal S, Koch F, Andrau JC, Winkler GS. The Ccr4-Not Deadenylase Subunits CNOT7 and CNOT8 Have Overlapping Roles and Modulate Cell Proliferation. Mol Biol Cell. 2009;20(17):3840-3850. [DOI:10.1091/mbc.e09-02-0146] [PMID] [PMCID]
12. Traven A, Hammet A, Tenis N, Denis CL, Heierhorst J. Ccr4-Not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae. Genetics. 2005:169(1): 65-75. [DOI:10.1534/genetics.104.030940] [PMID] [PMCID]
13. Mulder KW, Winkler GS, Timmers HT. DNA damage and replication stress induced transcription of RNR genes is dependent on the Ccr4-Not complex. Nucleic Acids Res. 2005;33(19):6384-6392. [DOI:10.1093/nar/gki938] [PMID] [PMCID]
14. Woolstencroft RN, Beilharz TH, Cook MA, Preiss T, Durocher D, Tyers M. Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length. J Cell Sci. 2006;119(Pt 24): 5178-92. [DOI:10.1242/jcs.03221] [PMID]
15. Yajima H, Lee KJ, Zhang S, Kobayashi J, Chen BP. DNA Double Strand Break Formation upon UV-Induced Replication Stress Activates ATM and DNA-PKcs Kinases. J Mol Biol. 2009:385(3):800-810. [DOI:10.1016/j.jmb.2008.11.036] [PMID] [PMCID]
16. Blackford AN, Jackson SP. ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell. 2017;66(6):801-817. [DOI:10.1016/j.molcel.2017.05.015] [PMID]
17. Shirai YT, Suzuki T, Morita M, Takahashi A, Yamamoto T. Multifunctional roles of the mammalian CCR4-NOT complex in physiological phenomena. Front Genet. 2014;5(286). [DOI:10.3389/fgene.2014.00286] [PMID] [PMCID]
18. Tucker M, Valencia-Sanchez MA, Staples RR, Chen J, Denis CL, Parker R. The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell. 2001;104(3):377-86. [DOI:10.1016/S0092-8674(01)00225-2]
19. Petit AP, Wohlbold L, Bawankar P, Huntzinger E, Schmidt S, Izaurralde E, et al. The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4-NOT deadenylase complex. Nucleic Acids Res. 2012;40(21):11058-72. [DOI:10.1093/nar/gks883] [PMID] [PMCID]
20. Tucker M, Staples RR, Valencia-Sanchez MA, Muhlrad D, Parker R. Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO J. 2002;21(6):1427-1436. [DOI:10.1093/emboj/21.6.1427] [PMID] [PMCID]
21. Temme C, Zhang L, Kremmer E, Ihling C, Chartier A, Sinz A, et al. Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation. RNA. 2010;16(7):1356-1370. [DOI:10.1261/rna.2145110] [PMID] [PMCID]
22. Schwede A, Ellis L, Luther J, Carrington M, Stoecklin G, Clayton C. A role for Caf1 in mRNA deadenylation and decay in trypanosomes and human cells. Nucleic Acids Res. 2008;36(10)3374-3388. [DOI:10.1093/nar/gkn108] [PMID] [PMCID]
23. Mostafa D, Takahashi A, Yanagiya A, Yamaguchi T, Abe T, Kureha T, et al. Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability. RNA Biol. 2020;17(3):403-416. [DOI:10.1080/15476286.2019.1709747] [PMID] [PMCID]
24. Maragozidis P, Karangeli M, Labrou M, Dimoulou G, Papaspyrou K, Salataj E, et al. Alterations of deadenylase expression in acute leukemias: evidence for poly(a)-specific ribonuclease as a potential biomarker. Acta Haematol. 2012;128(1):39-46. [DOI:10.1159/000337418] [PMID]
25. Takahashi S, Kontani, K, Araki, Y, Katada T. Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly. Nucleic Acids Res. 2007;35(4);1187-1197. [DOI:10.1093/nar/gkm015] [PMID] [PMCID]
26. Halazonetis TD, Shiloh Y. Many faces of ATM: eighth interna- tional workshop on ataxia-telangiectasia. Biochim Biophys Acta. 1999;1424(2-3):R45-55. [DOI:10.1016/S0304-419X(99)00023-2]
27. Giunta S, Belotserkovskaya R, Jackson SP. DNA damage signaling in response to double- strand breaks during mitosis. J Cell Biol. 2010: 190(2):197-207. [DOI:10.1083/jcb.200911156] [PMID] [PMCID]
28. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. 2000;10(15);886-95. [DOI:10.1016/S0960-9822(00)00610-2]
29. Schultz LB, Chehab NH, Malikzay A, Halazonetis TD. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol. 2000;151(7):1381-1390. [DOI:10.1083/jcb.151.7.1381] [PMID] [PMCID]
30. DiTullio RA, Mochan TA, Venere M, Bartkova J, Sehested M, Bartek J, et al. 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat Cell Biol. 2002;4(12)998-1002. [DOI:10.1038/ncb892] [PMID]
31. Glover L, Marques CA, Suska O, Horna D. Persistent DNA Damage Foci and DNA Replication with a Broken Chromosome in the African Trypanosome. mBio. 2019;10(4):e01252-19. [DOI:10.1128/mBio.01252-19] [PMID] [PMCID]
32. Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat Rev Mol Cell Biol. 2017;18(10):622-636. [DOI:10.1038/nrm.2017.67] [PMID] [PMCID]
33. Hustedt N, Durocher D. The control of DNA repair by the cell cycle. Nat Cell Biol. 2016;19(1)1-9. [DOI:10.1038/ncb3452] [PMID]
34. Cruz C, Castroviejo-Bermejo M, Gutie ́rrez-Enr ́ıquez M, Llop-Guevara A, Ibrahim YH, Gris-Oliver A, et al. RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer. Ann Oncol. 2018:29(5)1203-1210. [DOI:10.1093/annonc/mdy099] [PMID] [PMCID]

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