Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Autoři: Rudo Kieft aff001;  Yang Zhang aff001;  Alexandre P. Marand aff002;  Jose Dagoberto Moran aff001;  Robert Bridger aff001;  Lance Wells aff001;  Robert J. Schmitz aff002;  Robert Sabatini aff001
Působiště autorů: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America aff001;  Department of Genetics, University of Georgia, Athens, Georgia, United States of America aff002
Vyšlo v časopise: Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes. PLoS Genet 16(2): e32767. doi:10.1371/journal.pgen.1008390
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008390


Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3’-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.

Klíčová slova:

DNA transcription – DNA-binding proteins – Gene expression – Messenger RNA – RNA interference – Transcriptional control – Transcriptional termination – Trypanosoma brucei gambiense


1. Shandilya J, Roberts SG. The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling. Biochim Biophys Acta. 2012;1819(5):391–400. doi: 10.1016/j.bbagrm.2012.01.010 22306664

2. Kecman T, Kus K, Heo DH, Duckett K, Birot A, Liberatori S, et al. Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination. Cell Rep. 2018;25(1):259–69 e5. doi: 10.1016/j.celrep.2018.09.007 30282034

3. Schreieck A, Easter AD, Etzold S, Wiederhold K, Lidschreiber M, Cramer P, et al. RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7. Nat Struct Mol Biol. 2014;21(2):175–9. doi: 10.1038/nsmb.2753 24413056

4. Bollen M, Peti W, Ragusa MJ, Beullens M. The extended PP1 toolkit: designed to create specificity. Trends in biochemical sciences. 2010;35(8):450–8. doi: 10.1016/j.tibs.2010.03.002 20399103

5. Dancheck B, Nairn AC, Peti W. Detailed structural characterization of unbound protein phosphatase 1 inhibitors. Biochemistry. 2008;47(47):12346–56. doi: 10.1021/bi801308y 18954090

6. Ragusa MJ, Dancheck B, Critton DA, Nairn AC, Page R, Peti W. Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites. Nat Struct Mol Biol. 2010;17(4):459–64. doi: 10.1038/nsmb.1786 20305656

7. Jagiello I, Beullens M, Stalmans W, Bollen M. Subunit structure and regulation of protein phosphatase-1 in rat liver nuclei. J Biol Chem. 1995;270(29):17257–63. doi: 10.1074/jbc.270.29.17257 7615525

8. Kreivi JP, Trinkle-Mulcahy L, Lyon CE, Morrice NA, Cohen P, Lamond AI. Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity. FEBS Lett. 1997;420(1):57–62. doi: 10.1016/s0014-5793(97)01485-3 9450550

9. Lee JH, You J, Dobrota E, Skalnik DG. Identification and characterization of a novel human PP1 phosphatase complex. J Biol Chem. 2010;285(32):24466–76. doi: 10.1074/jbc.M110.109801 20516061

10. Lee JH, Skalnik DG. Wdr82 is a C-terminal domain-binding protein that recruits the Setd1A Histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes. Mol Cell Biol. 2008;28(2):609–18. doi: 10.1128/MCB.01356-07 17998332

11. Dichtl B, Blank D, Ohnacker M, Friedlein A, Roeder D, Langen H, et al. A role for SSU72 in balancing RNA polymerase II transcription elongation and termination. Mol Cell. 2002;10(5):1139–50. doi: 10.1016/s1097-2765(02)00707-4 12453421

12. He X, Khan AU, Cheng H, Pappas DL Jr., Hampsey M, Moore CL. Functional interactions between the transcription and mRNA 3' end processing machineries mediated by Ssu72 and Sub1. Genes Dev. 2003;17(8):1030–42. doi: 10.1101/gad.1075203 12704082

13. Vanoosthuyse V, Legros P, van der Sar SJ, Yvert G, Toda K, Le Bihan T, et al. CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation. PLoS genetics. 2014;10(6):e1004415. doi: 10.1371/journal.pgen.1004415 24945319

14. Nedea E, He X, Kim M, Pootoolal J, Zhong G, Canadien V, et al. Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'-ends. J Biol Chem. 2003;278(35):33000–10. doi: 10.1074/jbc.M304454200 12819204

15. Cheng H, He X, Moore C. The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3. Mol Cell Biol. 2004;24(7):2932–43. doi: 10.1128/MCB.24.7.2932-2943.2004 15024081

16. Nedea E, Nalbant D, Xia D, Theoharis NT, Suter B, Richardson CJ, et al. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes. Mol Cell. 2008;29(5):577–87. doi: 10.1016/j.molcel.2007.12.031 18342605

17. Austenaa LM, Barozzi I, Simonatto M, Masella S, Della Chiara G, Ghisletti S, et al. Transcription of Mammalian cis-Regulatory Elements Is Restrained by Actively Enforced Early Termination. Mol Cell. 2015;60(3):460–74. doi: 10.1016/j.molcel.2015.09.018 26593720

18. Clayton C, Michaeli S. 3' processing in protists. Wiley interdisciplinary reviews RNA. 2011;2(2):247–55. doi: 10.1002/wrna.49 21957009

19. Clayton CE. Gene expression in Kinetoplastids. Curr Opin Microbiol. 2016;32:46–51. doi: 10.1016/j.mib.2016.04.018 27177350

20. Michaeli S. Trans-splicing in trypanosomes: machinery and its impact on the parasite transcriptome. Future Microbiol. 2011;6(4):459–74. doi: 10.2217/fmb.11.20 21526946

21. Siegel TN, Gunasekera K, Cross GA, Ochsenreiter T. Gene expression in Trypanosoma brucei: lessons from high-throughput RNA sequencing. Trends Parasitol. 2011;27(10):434–41. doi: 10.1016/ 21737348

22. Borst P, Sabatini R. Base J: discovery, biosynthesis, and possible functions. Annu Rev Microbiol. 2008;62:235–51. doi: 10.1146/annurev.micro.62.081307.162750 18729733

23. Cliffe LJ, Siegel TN, Marshall M, Cross GA, Sabatini R. Two thymidine hydroxylases differentially regulate the formation of glucosylated DNA at regions flanking polymerase II polycistronic transcription units throughout the genome of Trypanosoma brucei. Nucleic Acids Res. 2010;38(12):3923–35. doi: 10.1093/nar/gkq146 20215442

24. Reynolds D, Hofmeister BT, Cliffe L, Alabady M, Siegel TN, Schmitz RJ, et al. Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei. PLoS genetics. 2016;12(1):e1005758. doi: 10.1371/journal.pgen.1005758 26796527

25. Siegel TN, Hekstra DR, Kemp LE, Figueiredo LM, Lowell JE, Fenyo D, et al. Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei. Genes Dev. 2009;23(9):1063–76. doi: 10.1101/gad.1790409 19369410

26. van Luenen HG, Farris C, Jan S, Genest PA, Tripathi P, Velds A, et al. Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania. Cell. 2012;150(5):909–21. doi: 10.1016/j.cell.2012.07.030 22939620

27. Reynolds D, Cliffe L, Forstner KU, Hon CC, Siegel TN, Sabatini R. Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei. Nucleic Acids Res. 2014;42(15):9717–29. doi: 10.1093/nar/gku714 25104019

28. Reynolds DL, Hofmeister BT, Cliffe L, Siegel TN, Anderson BA, Beverley SM, et al. Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination. Mol Microbiol. 2016;101(4):559–74. doi: 10.1111/mmi.13408 27125778

29. Schulz D, Zaringhalam M, Papavasiliou FN, Kim HS. Base J and H3.V Regulate Transcriptional Termination in Trypanosoma brucei. PLoS genetics. 2016;12(1):e1005762. doi: 10.1371/journal.pgen.1005762 26796638

30. Cliffe LJ, Hirsch G, Wang J, Ekanayake D, Bullard W, Hu M, et al. JBP1 and JBP2 Proteins Are Fe2+/2-Oxoglutarate-dependent Dioxygenases Regulating Hydroxylation of Thymidine Residues in Trypanosome DNA. J Biol Chem. 2012;287(24):19886–95. doi: 10.1074/jbc.M112.341974 22514282

31. Bullard W, da Rosa-Spiegler JL, Liu S, Wang D, Sabatini R. Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome. JBC. 2014.

32. Sekar A, Merritt C, Baugh L, Stuart K, Myler PJ. Tb927.10.6900 encodes the glucosyltransferase involved in synthesis of base J in Trypanosoma brucei. Mol Biochem Parasitol. 2014;196(1):9–11. doi: 10.1016/j.molbiopara.2014.07.005 25064607

33. Sabatini R, Cliffe L, Vainio L, Borst P. Enzymatic Formation of the Hypermodified DNA Base J. In: Grosjean H, editor. DNA and RNA Modification Enzymes: Comparative Structure, Mechanism, Function, Cellular Interactions and Evolution. Texas: Landes Biosciences; 2009. p. 120–31.

34. Cliffe LJ, Kieft R, Southern T, Birkeland SR, Marshall M, Sweeney K, et al. JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes. Nucleic Acids Res. 2009.

35. Kieft R, Brand V, Ekanayake DK, Sweeney K, DiPaolo C, Reznikoff WS, et al. JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei. Mol Biochem Parasitol. 2007;156(1):24–31. doi: 10.1016/j.molbiopara.2007.06.010 17706299

36. Vainio S, Genest PA, ter Riet B, van Luenen H, Borst P. Evidence that J-binding protein 2 is a thymidine hydroxylase catalyzing the first step in the biosynthesis of DNA base J. Mol Biochem Parasitol. 2009;164(2):157–61. doi: 10.1016/j.molbiopara.2008.12.001 19114062

37. Yu Z, Genest PA, ter Riet B, Sweeney K, DiPaolo C, Kieft R, et al. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase. Nucleic Acids Res. 2007;35(7):2107–15. doi: 10.1093/nar/gkm049 17389644

38. DiPaolo C, Kieft R, Cross M, Sabatini R. Regulation of trypanosome DNA glycosylation by a SWI2/SNF2-like protein. Mol Cell. 2005;17(3):441–51. doi: 10.1016/j.molcel.2004.12.022 15694344

39. Heidebrecht T, Christodoulou E, Chalmers MJ, Jan S, Ter Riet B, Grover RK, et al. The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1. Nucleic Acids Res. 2011;39(13):5715–28. doi: 10.1093/nar/gkr125 21415010

40. Sabatini R, Meeuwenoord N, van Boom JH, Borst P. Recognition of base J in duplex DNA by J-binding protein. J Biol Chem. 2002;277:958–66. doi: 10.1074/jbc.M109000200 11700315

41. Sabatini R, Meeuwenoord N, van Boom JH, Borst P. Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Journal of Biological Chemistry. 2002;277:28150–6. doi: 10.1074/jbc.M201487200 12029082

42. Bullard W, Cliffe L, Wang P, Wang Y, Sabatini R. Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA. Mol Biochem Parasitol. 2015;204(2):77–80. doi: 10.1016/j.molbiopara.2016.01.005 26815240

43. Bullard W, Kieft R, Sabatini R. A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA. Biol Methods Protoc. 2017;2(1).

44. van Leeuwen F, Kieft R, Cross M, Borst P. Biosynthesis and function of the modified DNA base beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei. Molecular & Cellular Biology. 1998;18(10):5643–51.

45. Aphasizhev R, Aphasizheva I, Nelson RE, Gao G, Simpson AM, Kang X, et al. Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria. The EMBO Journal. 2003;22(4):913–24. doi: 10.1093/emboj/cdg083 12574127

46. Hurley TD, Yang J, Zhang L, Goodwin KD, Zou Q, Cortese M, et al. Structural basis for regulation of protein phosphatase 1 by inhibitor-2. J Biol Chem. 2007;282(39):28874–83. doi: 10.1074/jbc.M703472200 17636256

47. Terrak M, Kerff F, Langsetmo K, Tao T, Dominguez R. Structural basis of protein phosphatase 1 regulation. Nature. 2004;429(6993):780–4. doi: 10.1038/nature02582 15164081

48. Jones DT, Cozzetto D. DISOPRED3: precise disordered region predictions with annotated protein-binding activity. Bioinformatics. 2015;31(6):857–63. doi: 10.1093/bioinformatics/btu744 25391399

49. Ward JJ, McGuffin LJ, Bryson K, Buxton BF, Jones DT. The DISOPRED server for the prediction of protein disorder. Bioinformatics. 2004;20(13):2138–9. doi: 10.1093/bioinformatics/bth195 15044227

50. Vacic V, Uversky VN, Dunker AK, Lonardi S. Composition Profiler: a tool for discovery and visualization of amino acid composition differences. BMC Bioinformatics. 2007;8:211. doi: 10.1186/1471-2105-8-211 17578581

51. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010;5(4):725–38. doi: 10.1038/nprot.2010.5 20360767

52. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods. 2015;12(1):7–8. doi: 10.1038/nmeth.3213 25549265

53. Brenchley R, Tariq H, McElhinney H, Szoor B, Huxley-Jones J, Stevens R, et al. The TriTryp phosphatome: analysis of the protein phosphatase catalytic domains. BMC Genomics. 2007;8:434. doi: 10.1186/1471-2164-8-434 18039372

54. Li Z, Tu X, Wang CC. Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei. Exp Cell Res. 2006;312(18):3504–16. doi: 10.1016/j.yexcr.2006.07.022 16949574

55. Schimanski B, Nguyen TN, Gunzl A. Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. Eukaryot Cell. 2005;4(11):1942–50. doi: 10.1128/EC.4.11.1942-1950.2005 16278461

56. Mellacheruvu D, Wright Z, Couzens AL, Lambert JP, St-Denis NA, Li T, et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods. 2013;10(8):730–6. doi: 10.1038/nmeth.2557 23921808

57. Reynolds D, Cliffe L, Sabatini R. 2-Oxoglutarate-dependent hydroxylases involved In DNA base J (β-D-Glucopyranosyloxymethyluracil) synthesis. In: Schofield CJ, Hausinger RP, editors. 2-Oxoglutarate-Dependent Oxygenases. Cambridge: Royal Society of Chemistry; 2015.

58. Goos C, Dejung M, Janzen CJ, Butter F, Kramer S. The nuclear proteome of Trypanosoma brucei. PLoS One. 2017;12(7):e0181884. doi: 10.1371/journal.pone.0181884 28727848

59. Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38(Database issue):D457–62. doi: 10.1093/nar/gkp851 19843604

60. Ciurciu A, Duncalf L, Jonchere V, Lansdale N, Vasieva O, Glenday P, et al. PNUTS/PP1 regulates RNAPII-mediated gene expression and is necessary for developmental growth. PLoS genetics. 2013;9(10):e1003885. doi: 10.1371/journal.pgen.1003885 24204300

61. Jerebtsova M, Klotchenko SA, Artamonova TO, Ammosova T, Washington K, Egorov VV, et al. Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1. Mol Cell Biochem. 2011;347(1–2):79–87. doi: 10.1007/s11010-010-0614-3 20941529

62. Huang G, Ulrich PN, Storey M, Johnson D, Tischer J, Tovar JA, et al. Proteomic analysis of the acidocalcisome, an organelle conserved from bacteria to human cells. PLoS Pathog. 2014;10(12):e1004555. doi: 10.1371/journal.ppat.1004555 25503798

63. Mayer A, di Iulio J, Maleri S, Eser U, Vierstra J, Reynolds A, et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell. 2015;161(3):541–54. doi: 10.1016/j.cell.2015.03.010 25910208

64. Neil H, Malabat C, d'Aubenton-Carafa Y, Xu Z, Steinmetz LM, Jacquier A. Widespread bidirectional promoters are the major source of cryptic transcripts in yeast. Nature. 2009;457(7232):1038–42. doi: 10.1038/nature07747 19169244

65. Nojima T, Gomes T, Grosso ARF, Kimura H, Dye MJ, Dhir S, et al. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing. Cell. 2015;161(3):526–40. doi: 10.1016/j.cell.2015.03.027 25910207

66. Preker P, Nielsen J, Kammler S, Lykke-Andersen S, Christensen MS, Mapendano CK, et al. RNA exosome depletion reveals transcription upstream of active human promoters. Science. 2008;322(5909):1851–4. doi: 10.1126/science.1164096 19056938

67. Xu Z, Wei W, Gagneur J, Perocchi F, Clauder-Munster S, Camblong J, et al. Bidirectional promoters generate pervasive transcription in yeast. Nature. 2009;457(7232):1033–7. doi: 10.1038/nature07728 19169243

68. Kolev NG, Franklin JB, Carmi S, Shi H, Michaeli S, Tschudi C. The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution. PLoS Pathog. 2010;6(9).

69. Wedel C, Forstner KU, Derr R, Siegel TN. GT-rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes. EMBO J. 2017;36(17):2581–94. doi: 10.15252/embj.201695323 28701485

70. Gros J, Kumar C, Lynch G, Yadav T, Whitehouse I, Remus D. Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA. Mol Cell. 2015;60(5):797–807. doi: 10.1016/j.molcel.2015.10.022 26656162

71. Mori S, Shirahige K. Perturbation of the activity of replication origin by meiosis-specific transcription. J Biol Chem. 2007;282(7):4447–52. doi: 10.1074/jbc.M609671200 17170106

72. Nieduszynski CA, Blow JJ, Donaldson AD. The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription. Nucleic Acids Res. 2005;33(8):2410–20. doi: 10.1093/nar/gki539 15860777

73. Nieduszynski CA, Knox Y, Donaldson AD. Genome-wide identification of replication origins in yeast by comparative genomics. Genes Dev. 2006;20(14):1874–9. doi: 10.1101/gad.385306 16847347

74. Snyder M, Sapolsky RJ, Davis RW. Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae. Mol Cell Biol. 1988;8(5):2184–94. doi: 10.1128/mcb.8.5.2184 3290652

75. Tiengwe C, Marcello L, Farr H, Dickens N, Kelly S, Swiderski M, et al. Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei. Cell Rep. 2012;2(1):185–97. doi: 10.1016/j.celrep.2012.06.007 22840408

76. Lombrana R, Alvarez A, Fernandez-Justel JM, Almeida R, Poza-Carrion C, Gomes F, et al. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major. Cell Rep. 2016;16(6):1774–86. doi: 10.1016/j.celrep.2016.07.007 27477279

77. Horn D. Antigenic variation in African trypanosomes. Mol Biochem Parasitol. 2014;195(2):123–9. doi: 10.1016/j.molbiopara.2014.05.001 24859277

78. Horn D, McCulloch R. Molecular mechanisms underlying the control of antigenic variation in African trypanosomes. Curr Opin Microbiol. 2010;13(6):700–5. doi: 10.1016/j.mib.2010.08.009 20884281

79. Muller LSM, Cosentino RO, Forstner KU, Guizetti J, Wedel C, Kaplan N, et al. Genome organization and DNA accessibility control antigenic variation in trypanosomes. Nature. 2018;563(7729):121–5. doi: 10.1038/s41586-018-0619-8 30333624

80. Cross GA, Kim HS, Wickstead B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol Biochem Parasitol. 2014;195(1):59–73. doi: 10.1016/j.molbiopara.2014.06.004 24992042

81. Wickstead B, Ersfeld K, Gull K. The small chromosomes of Trypanosoma brucei involved in antigenic variation are constructed around repetitive palindromes. Genome Res. 2004;14(6):1014–24. doi: 10.1101/gr.2227704 15173109

82. Hertz-Fowler C, Figueiredo LM, Quail MA, Becker M, Jackson A, Bason N, et al. Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS One. 2008;3(10):e3527. doi: 10.1371/journal.pone.0003527 18953401

83. Ehlers B, Czichos J, Overath P. RNA turnover in Trypanosoma brucei. Mol Cell Biol. 1987;7(3):1242–9. doi: 10.1128/mcb.7.3.1242 2436040

84. Kabiri M, Steverding D. Studies on the recycling of the transferrin receptor in Trypanosoma brucei using an inducible gene expression system. Eur J Biochem. 2000;267(11):3309–14. doi: 10.1046/j.1432-1327.2000.01361.x 10824117

85. Kerry LE, Pegg EE, Cameron DP, Budzak J, Poortinga G, Hannan KM, et al. Selective inhibition of RNA polymerase I transcription as a potential approach to treat African trypanosomiasis. PLoS Negl Trop Dis. 2017;11(3):e0005432. doi: 10.1371/journal.pntd.0005432 28263991

86. Vanhamme L, Berberof M, Le Ray D, Pays E. Stimuli of differentiation regulate RNA elongation in the transcription units for the major stage-specific antigens of Trypanosoma brucei. Nucleic Acids Res. 1995;23(11):1862–9. doi: 10.1093/nar/23.11.1862 7596810

87. Vanhamme L, Pays E, McCulloch R, Barry JD. An update on antigenic variation in African trypanosomes. TRENDS in Parasitology. 2001;17:338–43. doi: 10.1016/s1471-4922(01)01922-5 11423377

88. Vanhamme L, Poelvoorde P, Pays A, Tebabi P, Van Xong H, Pays E. Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei. Mol Microbiol. 2000;36(2):328–40. doi: 10.1046/j.1365-2958.2000.01844.x 10792720

89. Clayton C, Shapira M. Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. Mol Biochem Parasitol. 2007;156(2):93–101. doi: 10.1016/j.molbiopara.2007.07.007 17765983

90. Kelly S, Kramer S, Schwede A, Maini PK, Gull K, Carrington M. Genome organization is a major component of gene expression control in response to stress and during the cell division cycle in trypanosomes. Open Biol. 2012;2(4):120033. doi: 10.1098/rsob.120033 22724062

91. Engstler M, Boshart M. Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei. Genes Dev. 2004;18(22):2798–811. doi: 10.1101/gad.323404 15545633

92. Rolin S, Hancocq-Quertier J, Paturiaux-Hanocq F, Nolan DP, Pays E. Mild acid stress as a differentiation trigger in Trypanosoma brucei. Mol Biochem Parasitol. 1998;93(2):251–62. doi: 10.1016/s0166-6851(98)00046-2 9662709

93. Biebinger S, Rettenmaier S, Flaspohler J, Hartmann C, Pena-Diaz J, Wirtz LE, et al. The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context. Nucleic Acids Research. 1996;24(7):1202–11. doi: 10.1093/nar/24.7.1202 8614620

94. Ruthenburg AJ, Wang W, Graybosch DM, Li H, Allis CD, Patel DJ, et al. Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex. Nat Struct Mol Biol. 2006;13(8):704–12. doi: 10.1038/nsmb1119 16829959

95. Soares LM, Buratowski S. Yeast Swd2 is essential because of antagonism between Set1 histone methyltransferase complex and APT (associated with Pta1) termination factor. J Biol Chem. 2012;287(19):15219–31. doi: 10.1074/jbc.M112.341412 22431730

96. Ng HH, Robert F, Young RA, Struhl K. Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol Cell. 2003;11(3):709–19. doi: 10.1016/s1097-2765(03)00092-3 12667453

97. Dingar D, Tu WB, Resetca D, Lourenco C, Tamachi A, De Melo J, et al. MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability. Nature communications. 2018;9(1):3502. doi: 10.1038/s41467-018-05660-0 30158517

98. Parua PK, Booth GT, Sanso M, Benjamin B, Tanny JC, Lis JT, et al. A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II. Nature. 2018;558(7710):460–4. doi: 10.1038/s41586-018-0214-z 29899453

99. Srivastava A, Badjatia N, Lee JH, Hao B, Gunzl A. An RNA polymerase II-associated TFIIF-like complex is indispensable for SL RNA gene transcription in Trypanosoma brucei. Nucleic Acids Res. 2017.

100. Urbaniak MD, Martin DM, Ferguson MA. Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei. J Proteome Res. 2013;12(5):2233–44. doi: 10.1021/pr400086y 23485197

101. Guo Z, Stiller JW. Comparative genomics of cyclin-dependent kinases suggest co-evolution of the RNAP II C-terminal domain and CTD-directed CDKs. BMC Genomics. 2004;5:69. doi: 10.1186/1471-2164-5-69 15380029

102. Das A, Banday M, Fisher MA, Chang YJ, Rosenfeld J, Bellofatto V. An essential domain of an early-diverged RNA polymerase II functions to accurately decode a primitive chromatin landscape. Nucleic Acids Res. 2017;45(13):7886–96. doi: 10.1093/nar/gkx486 28575287

103. Das A, Bellofatto V. The non-canonical CTD of RNAP-II is essential for productive RNA synthesis in Trypanosoma brucei. PLoS One. 2009;4(9):e6959. doi: 10.1371/journal.pone.0006959 19742309

104. Rocha AA, Moretti NS, Schenkman S. Stress induces changes in the phosphorylation of Trypanosoma cruzi RNA polymerase II, affecting its association with chromatin and RNA processing. Eukaryot Cell. 2014;13(7):855–65. doi: 10.1128/EC.00066-14 24813189

105. Core LJ, Waterfall JJ, Lis JT. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. 2008;322(5909):1845–8. doi: 10.1126/science.1162228 19056941

106. Preker P, Nielsen J, Schierup MH, Jensen TH. RNA polymerase plays both sides: vivid and bidirectional transcription around and upstream of active promoters. Cell Cycle. 2009;8(8):1106–7. 19305135

107. Sigova AA, Mullen AC, Molinie B, Gupta S, Orlando DA, Guenther MG, et al. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc Natl Acad Sci U S A. 2013;110(8):2876–81. doi: 10.1073/pnas.1221904110 23382218

108. Wei W, Pelechano V, Jarvelin AI, Steinmetz LM. Functional consequences of bidirectional promoters. Trends Genet. 2011;27(7):267–76. doi: 10.1016/j.tig.2011.04.002 21601935

109. Flynn RA, Almada AE, Zamudio JR, Sharp PA. Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome. Proc Natl Acad Sci U S A. 2011;108(26):10460–5. doi: 10.1073/pnas.1106630108 21670248

110. Ntini E, Jarvelin AI, Bornholdt J, Chen Y, Boyd M, Jorgensen M, et al. Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality. Nat Struct Mol Biol. 2013;20(8):923–8. doi: 10.1038/nsmb.2640 23851456

111. Schulz D, Schwalb B, Kiesel A, Baejen C, Torkler P, Gagneur J, et al. Transcriptome surveillance by selective termination of noncoding RNA synthesis. Cell. 2013;155(5):1075–87. doi: 10.1016/j.cell.2013.10.024 24210918

112. Descostes N, Heidemann M, Spinelli L, Schuller R, Maqbool MA, Fenouil R, et al. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells. Elife. 2014;3:e02105. doi: 10.7554/eLife.02105 24842994

113. Fong N, Saldi T, Sheridan RM, Cortazar MA, Bentley DL. RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction. Mol Cell. 2017;66(4):546–57 e3. doi: 10.1016/j.molcel.2017.04.016 28506463

114. Shah N, Maqbool MA, Yahia Y, El Aabidine AZ, Esnault C, Forne I, et al. Tyrosine-1 of RNA Polymerase II CTD Controls Global Termination of Gene Transcription in Mammals. Mol Cell. 2018;69(1):48–61 e6. doi: 10.1016/j.molcel.2017.12.009 29304333

115. Shetty A, Kallgren SP, Demel C, Maier KC, Spatt D, Alver BH, et al. Spt5 Plays Vital Roles in the Control of Sense and Antisense Transcription Elongation. Mol Cell. 2017;66(1):77–88 e5. doi: 10.1016/j.molcel.2017.02.023 28366642

116. Dellino GI, Cittaro D, Piccioni R, Luzi L, Banfi S, Segalla S, et al. Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing. Genome Res. 2013;23(1):1–11. doi: 10.1101/gr.142331.112 23187890

117. Miotto B, Ji Z, Struhl K. Selectivity of ORC binding sites and the relation to replication timing, fragile sites, and deletions in cancers. Proc Natl Acad Sci U S A. 2016;113(33):E4810–9. doi: 10.1073/pnas.1609060113 27436900

118. Soudet J, Gill JK, Stutz F. Noncoding transcription influences the replication initiation program through chromatin regulation. Genome Res. 2018;28(12):1882–93. doi: 10.1101/gr.239582.118 30401734

119. Kim HS. Genome-wide function of MCM-BP in Trypanosoma brucei DNA replication and transcription. Nucleic Acids Res. 2019;47(2):634–47. doi: 10.1093/nar/gky1088 30407533

120. Benmerzouga I, Concepcion-Acevedo J, Kim HS, Vandoros AV, Cross GA, Klingbeil MM, et al. Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching. Mol Microbiol. 2013;87(1):196–210. doi: 10.1111/mmi.12093 23216794

121. Anderson SJ, Sikes ML, Zhang Y, French SL, Salgia S, Beyer AL, et al. The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively. J Biol Chem. 2011;286(21):18816–24. doi: 10.1074/jbc.M110.202101 21467039

122. Viktorovskaya OV, Appling FD, Schneider DA. Yeast transcription elongation factor Spt5 associates with RNA polymerase I and RNA polymerase II directly. J Biol Chem. 2011;286(21):18825–33. doi: 10.1074/jbc.M110.202119 21467036

123. Wirtz E, Leal S, Ochatt C, Cross GA. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol. 1999;99(1):89–101. doi: 10.1016/s0166-6851(99)00002-x 10215027

124. Wickstead B, Ersfeld K, Gull K. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol Biochem Parasitol. 2002;125(1–2):211–6. doi: 10.1016/s0166-6851(02)00238-4 12467990

125. Oberholzer M, Morand S, Kunz S, Seebeck T. A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes. Mol Biochem Parasitol. 2006;145(1):117–20. doi: 10.1016/j.molbiopara.2005.09.002 16269191

126. van Leeuwen F, Wijsman ER, Kieft R, van der Marel GA, van Boom JH, Borst P. Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei. Genes & Development. 1997;11(23):3232–41.

127. Lim JM, Sherling D, Teo CF, Hausman DB, Lin D, Wells L. Defining the regulated secreted proteome of rodent adipocytes upon the induction of insulin resistance. J Proteome Res. 2008;7(3):1251–63. doi: 10.1021/pr7006945 18237111

128. Baez-Santos YM, Mielech AM, Deng X, Baker S, Mesecar AD. Catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the Middle East respiratory syndrome coronavirus. J Virol. 2014;88(21):12511–27. doi: 10.1128/JVI.01294-14 25142582

129. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884–i90. doi: 10.1093/bioinformatics/bty560 30423086

130. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods. 2012;9(4):357–9. doi: 10.1038/nmeth.1923 22388286

131. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. doi: 10.1093/bioinformatics/btp352 19505943

132. UniProt C. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019;47(D1):D506–D15. doi: 10.1093/nar/gky1049 30395287

133. Potter SC, Luciani A, Eddy SR, Park Y, Lopez R, Finn RD. HMMER web server: 2018 update. Nucleic Acids Res. 2018;46(W1):W200–W4. doi: 10.1093/nar/gky448 29905871

134. Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–3. doi: 10.1093/bioinformatics/btp348 19505945

135. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3. doi: 10.1093/bioinformatics/btu033 24451623

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 2
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy Podcasty Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

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