RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis

Autoři: Gera A. Pavlova aff001;  Julia V. Popova aff001;  Evgeniya N. Andreyeva aff001;  Lyubov A. Yarinich aff001;  Mikhail O. Lebedev aff001;  Alyona V. Razuvaeva aff001;  Tatiana D. Dubatolova aff001;  Anastasiya L. Oshchepkova aff001;  Claudia Pellacani aff005;  Maria Patrizia Somma aff005;  Alexey V. Pindyurin aff001;  Maurizio Gatti aff005
Působiště autorů: Institute of Molecular and Cellular Biology, Siberian Branch of RAS, Novosibirsk, Russia aff001;  Institute of Cytology and Genetics, Siberian Branch of RAS, Novosibirsk, Russia aff002;  Novosibirsk State University, Novosibirsk, Russia aff003;  Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS, Novosibirsk, Russia aff004;  IBPM CNR c/o Department of Biology and Biotechnology, Sapienza University of Rome, Rome, Italy aff005
Vyšlo v časopise: RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis. PLoS Genet 15(9): e32767. doi:10.1371/journal.pgen.1008371
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008371


The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.

Klíčová slova:

Biology and life sciences – Genetics – Epigenetics – RNA interference – Gene expression – DNA transcription – Genetic interference – Biochemistry – Nucleic acids – RNA – Cell biology – Cellular structures and organelles – Centrosomes – Centrioles – Cell processes – Cell cycle and cell division – Telophase – Mitosis – Metaphase – Chromosome biology – Organisms – Eukaryota – Animals – Invertebrates – Arthropoda – Insects – Drosophila – Drosophila melanogaster – Research and analysis methods – Animal studies – Experimental organism systems – Model organisms – Animal models


1. Yokoyama H, Gruss OJ. New mitotic regulators released from chromatin. Front Oncol. 2013;3:308. doi: 10.3389/fonc.2013.00308 24380075

2. Ravens S, Fournier M, Ye T, Stierle M, Dembele D, Chavant V, et al. Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation. Elife. 2014;3:e02104

3. Chelmicki T, Dündar F, Turley MJ, Khanam T, Aktas T, Ramírez F, et al. MOF-associated complexes ensure stem cell identity and Xist repression. Elife. 2014;3:e02024. doi: 10.7554/eLife.02024 24842875

4. Koolen DA, Kramer JM, Neveling K, Nillesen WM, Moore-Barton HL, Elmslie FV, et al. Mutations in the chromatin modifier gene KANSL1 cause the 17q21.31 microdeletion syndrome. Nat Genet. 2012; 44(6):639–641. doi: 10.1038/ng.2262 22544363

5. Zollino M, Orteschi D, Murdolo M, Lattante S, Battaglia D, Stefanini C, et al. Mutations in KANSL1 cause the 17q21.31 microdeletion syndrome phenotype. Nat Genet. 2012;44(6):636–638. doi: 10.1038/ng.2257 22544367

6. Gilissen C, Hehir-Kwa JY, Thung DT, van de Vorst M, van Bon BWM, Willemsen MH, et al. Genome sequencing identifies major causes of severe intellectual disability. Nature. 2014;511(7509):344–347. doi: 10.1038/nature13394 24896178

7. Meunier S, Vernos I. K-fibre minus ends are stabilized by a RanGTP-dependent mechanism essential for functional spindle assembly. Nat Cell Biol. 2011;13(12):1406–1414. doi: 10.1038/ncb2372 22081094

8. Meunier S, Shvedunova M, Van Nguyen N, Avila L, Vernos I, Akhtar A. An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis. Nat Commun. 2015;6:7889. doi: 10.1038/ncomms8889 26243146

9. Ali A, Veeranki SN, Chinchole A, Tyagi S. MLL/WDR5 complex regulates Kif2A localization to ensure chromosome congression and proper spindle assembly during mitosis. Dev Cell. 2017;41(6):605–622.e7. doi: 10.1016/j.devcel.2017.05.023 28633016

10. Raja SJ, Charapitsa I, Conrad T, Vaquerizas JM, Gebhardt P, Holz H, et al. The nonspecific lethal complex is a transcriptional regulator in Drosophila. Mol Cell. 2010;38(6):827–841. doi: 10.1016/j.molcel.2010.05.021 20620954

11. Pascual-Garcia P, Jeong J, Capelson M. Nucleoporin Nup98 associates with Trx/MLL and NSL histone-modifying complexes and regulates Hox gene expression. Cell Rep. 2014;9(2):433–442. doi: 10.1016/j.celrep.2014.09.002 25310983

12. Feller C, Prestel M, Hartmann H, Straub T, Söding J, Becker PB. The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset. Nucleic Acids Res. 2012;40(4):1509–1522. doi: 10.1093/nar/gkr869 22039099

13. Dias J, Van Nguyen N, Georgiev P, Gaub A, Brettschneider J, Cusack S, et al. Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex. Genes Dev. 2014;28(9):929–942. doi: 10.1101/gad.240200.114 24788516

14. Lam KC, Mühlpfordt F, Vaquerizas JM, Raja SJ, Holz H, Luscombe NM, et al. The NSL complex regulates housekeeping genes in Drosophila. PLoS Genet. 2012;8(6):e1002736. doi: 10.1371/journal.pgen.1002736 22723752

15. Goshima G, Wollman R, Goodwin SS, Zhang N, Scholey JM, Vale RD, et al. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science. 2007;316(5823):417–421. doi: 10.1126/science.1141314 17412918

16. Dobbelaere J, Josué F, Suijkerbuijk S, Baum B, Tapon N, Raff J. A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol. 2008;6(9):e224. doi: 10.1371/journal.pbio.0060224 18798690

17. Somma MP, Ceprani F, Bucciarelli E, Naim V, De Arcangelis V, Piergentili R, et al. Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference. PLoS Genet. 2008;4(7):e1000126. doi: 10.1371/journal.pgen.1000126 18797514

18. Blower MD, Karpen GH. The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions. Nat Cell Biol. 2001;3(8):730–739. doi: 10.1038/35087045 11483958

19. Pesenti ME, Weir JR, Musacchio A. Progress in the structural and functional characterization of kinetochores. Curr Opin Struct Biol. 2016;37:152–163. doi: 10.1016/j.sbi.2016.03.003 27039078

20. Dix CI, Raff JW. Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication. Curr Biol. 2007;17(20):1759–1764. doi: 10.1016/j.cub.2007.08.065 17919907

21. Giansanti MG, Bucciarelli E, Bonaccorsi S, Gatti M. Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation. Curr Biol. 2008;18(4):303–309. doi: 10.1016/j.cub.2008.01.058 18291647

22. Lee H, McManus CJ, Cho D-Y, Eaton M, Renda F, Somma MP, et al. DNA copy number evolution in Drosophila cell lines. Genome Biol. 2014;15(8):R70. doi: 10.1186/gb-2014-15-8-r70 25262759

23. Bettencourt-Dias M, Rodrigues-Martins A, Carpenter L, Riparbelli M, Lehmann L, Gatt MK, et al. SAK/PLK4 is required for centriole duplication and flagella development. Curr Biol. 2005;15(24):2199–2207. doi: 10.1016/j.cub.2005.11.042 16326102

24. Laycock JE, Savoian MS, Glover DM. Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo. J Cell Sci. 2006;119(Pt 11):2354–2361. doi: 10.1242/jcs.02957 16723741

25. Varmark H, Llamazares S, Rebollo E, Lange B, Reina J, Schwarz H, et al. Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila. Curr Biol. 2007;17(20):1735–1745. doi: 10.1016/j.cub.2007.09.031 17935995

26. Dzhindzhev NS, Yu QD, Weiskopf K, Tzolovsky G, Cunha-Ferreira I, Riparbelli M, et al. Asterless is a scaffold for the onset of centriole assembly. Nature. 2010;467(7316):714–718. doi: 10.1038/nature09445 20852615

27. Mennella V, Keszthelyi B, McDonald KL, Chhun B, Kan F, Rogers GC, et al. Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat Cell Biol. 2012;14(11):1159–1168. doi: 10.1038/ncb2597 23086239

28. Pellacani C, Bucciarelli E, Renda F, Hayward D, Palena A, Chen J, et al. Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation. Elife. 2018;7:e40325 doi: 10.7554/eLife.40325 30475206

29. Bonaccorsi S, Giansanti MG, Gatti M. Spindle assembly in Drosophila neuroblasts and ganglion mother cells. Nat Cell Biol. 2000;2(1):54–56. doi: 10.1038/71378 10620808

30. Megraw TL, Kao L-R, Kaufman TC. Zygotic development without functional mitotic centrosomes. Curr Biol. 2001;11(2):116–120. doi: 10.1016/s0960-9822(01)00017-3 11231128

31. Basto R, Lau J, Vinogradova T, Gardiol A, Woods CG, Khodjakov A, et al. Flies without centrioles. Cell. 2006;125(7):1375–1386. doi: 10.1016/j.cell.2006.05.025 16814722

32. Renda F, Pellacani C, Strunov A, Bucciarelli E, Naim V, Bosso G, et al. The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure. PLoS Genet. 2017;13(5):e1006784. doi: 10.1371/journal.pgen.1006784 28505193

33. Maiato H, Sampaio P, Lemos CL, Findlay J, Carmena M, Earnshaw WC, et al. MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity. J Cell Biol. 2002;157(5):749–760. doi: 10.1083/jcb.200201101 12034769

34. Przewloka MR, Zhang W, Costa P, Archambault V, D’Avino PP, Lilley KS, et al. Molecular analysis of core kinetochore composition and assembly in Drosophila melanogaster. PLoS ONE. 2007;2(5):e478. doi: 10.1371/journal.pone.0000478 17534428

35. Schittenhelm RB, Heeger S, Althoff F, Walter A, Heidmann S, Mechtler K, et al. Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma. 2007;116(4):385–402. doi: 10.1007/s00412-007-0103-y 17333235

36. Liu Y, Petrovic A, Rombaut P, Mosalaganti S, Keller J, Raunser S, et al. Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster. Open Biol. 2016;6(2):150236. doi: 10.1098/rsob.150236 26911624

37. Hu C-K, Coughlin M, Mitchison TJ. Midbody assembly and its regulation during cytokinesis. Mol Biol Cell. 2012;23(6):1024–1034. doi: 10.1091/mbc.E11-08-0721 22278743

38. Palozola KC, Lerner J, Zaret KS. A changing paradigm of transcriptional memory propagation through mitosis. Nat Rev Mol Cell Biol. 2019;20(1):55–64. doi: 10.1038/s41580-018-0077-z 30420736

39. DeLuca JG, Musacchio A. Structural organization of the kinetochore–microtubule interface. Curr Opin Cell Biol. 2012;24(1):48–56. doi: 10.1016/j.ceb.2011.11.003 22154944

40. Stevens NR, Roque H, Raff JW. DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement. Dev Cell. 2010;19(6):913–919. doi: 10.1016/j.devcel.2010.11.010 21145506

41. Arquint C, Nigg EA. The PLK4-STIL-SAS-6 module at the core of centriole duplication. Biochem Soc Trans. 2016;44(5):1253–1263. doi: 10.1042/BST20160116 27911707

42. Williams B, Leung G, Maiato H, Wong A, Li Z, Williams EV, et al. Mitch–a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation in Drosophila. J Cell Sci. 2007;120(Pt 20):3522–3533. doi: 10.1242/jcs.012112 17895365

43. Novak ZA, Conduit PT, Wainman A, Raff JW. Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos. Curr Biol. 2014;24(11):1276–1282. doi: 10.1016/j.cub.2014.04.023 24835456

44. Conduit PT, Wainman A, Raff JW. Centrosome function and assembly in animal cells. Nat Rev Mol Cell Biol. 2015;16(10):611–624. doi: 10.1038/nrm4062 26373263

45. Zurita M, Merino C. The transcriptional complexity of the TFIIH complex. Trends Genet. 2003;19(10):578–584. doi: 10.1016/j.tig.2003.08.005 14550632

46. Compe E, Egly J-M. Nucleotide excision repair and transcriptional regulation: TFIIH and Beyond. Annu Rev Biochem. 2016;85:265–290. doi: 10.1146/annurev-biochem-060815-014857 27294439

47. Fregoso M, Lainé J-P, Aguilar-Fuentes J, Mocquet V, Reynaud E, Coin F, et al. DNA repair and transcriptional deficiencies caused by mutations in the Drosophila p52 subunit of TFIIH generate developmental defects and chromosome fragility. Mol Cell Biol. 2007;27(10):3640–3650. doi: 10.1128/MCB.00030-07 17339330

48. Cruz-Becerra G, Valerio-Cabrera S, Juárez M, Bucio-Mendez A, Zurita M. TFIIH localization is highly dynamic during zygotic genome activation in Drosophila, and its depletion causes catastrophic mitosis. J Cell Sci. 2018;131(9): jcs.211631.

49. Ito S, Tan LJ, Andoh D, Narita T, Seki M, Hirano Y, et al. MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation. Mol Cell. 2010;39(4):632–640. doi: 10.1016/j.molcel.2010.07.029 20797633

50. Rambout X, Detiffe C, Bruyr J, Mariavelle E, Cherkaoui M, Brohée S, et al. The transcription factor ERG recruits CCR4–NOT to control mRNA decay and mitotic progression. Nat Struct Mol Biol. 2016;23(7):663–672. doi: 10.1038/nsmb.3243 27273514

51. Bailey JK, Fields AT, Cheng K, Lee A, Wagenaar E, Lagrois R, et al. WD repeat-containing protein 5 (WDR5) localizes to the midbody and regulates abscission. J Biol Chem. 2015;290(14):8987–9001. doi: 10.1074/jbc.M114.623611 25666610

52. Andersen JS, Wilkinson CJ, Mayor T, Mortensen P, Nigg EA, Mann M. Proteomic characterization of the human centrosome by protein correlation profiling. Nature. 2003;426(6966):570–574. doi: 10.1038/nature02166 14654843

53. Müller H, Schmidt D, Steinbrink S, Mirgorodskaya E, Lehmann V, Habermann K, et al. Proteomic and functional analysis of the mitotic Drosophila centrosome. EMBO J. 2010;29(19):3344–3357. doi: 10.1038/emboj.2010.210 20818332

54. Echard A, Hickson GRX, Foley E, O’Farrell PH. Terminal cytokinesis events uncovered after an RNAi screen. Curr Biol. 2004;14(18):1685–1693. doi: 10.1016/j.cub.2004.08.063 15380073

55. Eggert US, Kiger AA, Richter C, Perlman ZE, Perrimon N, Mitchison TJ, et al. Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biol. 2004;2(12):e379. doi: 10.1371/journal.pbio.0020379 15547975

56. Skop AR, Liu H, Yates J III, Meyer BJ, Heald R. Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms. Science. 2004;305(5680):61–66. doi: 10.1126/science.1097931 15166316

57. Fares MA. The evolution of protein moonlighting: adaptive traps and promiscuity in the chaperonins. Biochem Soc Trans. 2014;42(6):1709–1714. doi: 10.1042/BST20140225 25399594

58. Copley SD. An evolutionary perspective on protein moonlighting. Biochem Soc Trans. 2014;42(6):1684–1691. doi: 10.1042/BST20140245 25399590

59. Gottesfeld JM, Forbes DJ. Mitotic repression of the transcriptional machinery. Trends Biochem Sci. 1997;22(6):197–202. doi: 10.1016/s0968-0004(97)01045-1 9204705

60. Palozola KC, Liu H, Nicetto D, Zaret KS. Low-level, global transcription during mitosis and dynamic gene reactivation during mitotic exit. Cold Spring Harb Symp Quant Biol. 2017;82:197–205. doi: 10.1101/sqb.2017.82.034280 29348325

61. Somma MP, Fasulo B, Cenci G, Cundari E, Gatti M. Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol Biol Cell. 2002;13(7):2448–2460. doi: 10.1091/mbc.01-12-0589 12134082

62. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622. doi: 10.1373/clinchem.2008.112797 19246619

63. Chalkley GE, Verrijzer CP. Immuno-depletion and purification strategies to study chromatin-remodeling factors in vitro. Methods Enzymol. 2004;377:421–442. doi: 10.1016/S0076-6879(03)77028-1 14979043

64. Lehner CF, O’Farrell PH. The roles of Drosophila Cyclins A and B in mitotic control. Cell. 1990;61(3):535–547. doi: 10.1016/0092-8674(90)90535-m 2139805

65. Neph S, Kuehn MS, Reynolds AP, Haugen E, Thurman RE, Johnson AK, et al. BEDOPS: high-performance genomic feature operations. Bioinformatics. 2012;28(14):1919–1920. doi: 10.1093/bioinformatics/bts277 22576172

Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics

2019 Číslo 9

Nejčtenější v tomto čísle

Zvyšte si kvalifikaci online z pohodlí domova

Třikrát z interní medicíny
nový kurz
Autoři: MUDr. Jana Kubátová

Pokročilá Parkinsonova nemoc − úskalí a možnosti léčby
Autoři: doc. MUDr. Marek Baláž, Ph.D.

Léčba diabetes mellitus 2. typu pomocí GLP- 1 RA

Depresivní porucha a zánětlivé procesy
Autoři: MUDr. Juraj Tkáč

Methotrexát a jeho formy podávání v revmatologii
Autoři: MUDr. Liliana Šedová

Všechny kurzy
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se