Роль системы РНК-интерференции в регуляции длины теломер Drosophila тема диссертации и автореферата по ВАК РФ 03.00.03, кандидат биологических наук Квон, Дмитрий Аркадьевич

• Квон, Дмитрий Аркадьевич
• кандидат биологических наук
• 2008

Оглавление диссертации кандидат биологических наук Квон, Дмитрий Аркадьевич

СПИСОК СОКРАЩЕНИЙ

ОБЗОР ЛИТЕРАТУРЫ

Введение

Принципы организации теломер, образуемых теломеразой

Теломераза - фермент, поддерживающий длину теломер

Теломеразная РНК, ее взаимодействие с белками теломеразного комплекса

Белковые компоненты теломерного комплекса

TRF-белки -основные структурные белковые компоненты теломеры

Другие компоненты теломерного белкового комплекса

Принципы оранизации теломер Drosophila

Структура теломер Drosophila

Белковые компоненты теломер Drosophila

Регуляция транскрипции теломерных ретротранспозонов 15 Система РНК интерференции и ее роль в регуляции экспрессии ретротранспозонов

Общие принципы работы системы РНКи

Роль РНКи в регуляции экспрессии ретротранспозонов

МАТЕРИАЛЫ И МЕТОДЫ

Лабораторные линии, использованные в работе

Выделение геномной ДНК

ПЦР на геномной ДНК

Полукличественная ПЦР на геномной ДНК

Southern-блот анализ.

Рестрикция и гель-электрофорез.

Синтез РНК зонда для последующей гибридизации.

Гибридизация с меченым зондом.

Клонирование ПЦР-продукта.

Лигирование.

Приготовление компетентных клеток Е. coli.

Трансформация клеток E.coli и высев на селективную среду.

Выделение плазмидной ДНК

Секвенирование.

Меченые праймера.

ПЦР с термосеквеназой. 33 Клонирование промоторных областей рстротранспозонов

НеТ-А и TAHRE для измерения их активности 33 Трансфекция культуры клеток Drosophila и количественное определение активности галактозидазы

Выделение тотальной РНК Drosophila

RT-PCR (ОТ-ПЦР)

РНК in situ гибридизация

Фиксация яичников Drosophila

Получение зонда

Гибридизация

Детекция

РЕЗУЛЬТАТЫ 38 Сравнение экспрессии ретротранспозонов НеТ-А и

TART в яичниках мух, мутантных по генам системы РНКи.

Описание генетической системы, использованной в работе 40 Мутации генов spn-E и aub приводят к повышенной частоте транспозиций теломерных ретроэлементов к концу хромосомы 41 Мутации генов spn-E и aub в гетерозиготном состоянии вызывают активные транспозиции на конец хромосомы ретротранспозона TART 43 Активные транспозиции ретротранспозона НеТ-А к концу хромосомы происходят на фоне мутации гена spn-E в гомозиготном состоянии 48 ОТ-ПЦР анализ экспрессии НеТ-А и TART в яичниках мутантов по гену spn-E.

Ретротранспозон TAHRE участвует в поддержании теломер Drosophila. 51 Ретротранспозон TAHRE присутствует в геномах разных линий D. melanogaster и других видов Drosophila

3' область TAHRE обладает промоторной активностью

ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ

ВЫВОДЫ

Заключение диссертации по теме «Молекулярная биология», Квон, Дмитрий Аркадьевич

Выводы:

1. Транскрипты теломерных ретротранспозонов НеТ-А и TART накапливаются в яичниках мух, мутантных по генам РНКи spn-E и aub. В отличие от транскриптов НеТ-А, локализующихся в ооците, транскрипты TART накапливаются на поздних стадиях оогенеза в питающих клетках.

2. Продемонстрировано участие механизма РНКи в контроле длины теломер дрозофилы. Частота транспозиций теломерных ретротранспозонов к концу терминально-делетированной хромосомы увеличивается на фоне мутаций РНКи генов spn-E и aub. Мутантный аллель в гетерозиготном состоянии вызывает преимущественные транспозиции ретроэлемента TART, а в гомозиготном - НеТ-А элемента.

3. Недавно обнаруженный ретротранспозон TAHRE способен перемещаться на конец хромосомы, и, следовательно, этот элемент является полноправным участником поддержания теломер у дрозофилы.

4. Промотор ретротранспозона TAHRE, так же как у НеТ-А, находится в конце 3' нетранслируемой области данного элемента.

Список литературы диссертационного исследования кандидат биологических наук Квон, Дмитрий Аркадьевич, 2008 год

1. Кленов М.С., Гвоздев В.А. 2005. Формирование гетерохроматина: роль коротких РНК иметилирования ДНК. Биохимия 70,11: 1445- 459 Котельников Р.Н.,.Шпиз С.Г, Калмыкова А.И.,.Гвоздев В.А. 2006. Белки, связывающие

2. РНК, в процессах РНК-интерференции. Молекулярная биология 40, 4:595-608 Оловников A.M. Принцип маргинотомии в матричном синтезе полинуклеотидов. 1971.

3. Доклады АН СССР. 201: 1496-1499. Щербакова Д.М., Зверева М.Е., Шпанченко О.В.Донцова О.А. 2006. Теломераза: строение и свойства фермента, особенности теломеразы дрожжей. Биохимия 40:580-584

4. Aravin, A.A., M.S. Klenov, Y.V. Vagin, F. Bantignies, G. Cavalli, and V.A. Gvozdev. 2004.

5. Baulcombe, D. 2004. RNA silencing in plants. Nature 431: 356-63.

6. Baumann, P. and T.R. Cech. 2000. Protection of telomeres by the Ku protein in fission yeast.

7. Mol Biol Cell 11:3265-75. Bi, X., D. Srikanta, L. Fanti, S. Pimpinelli, R. Badugu, R. Kellum, and Y.S. Rong. 2005.

8. Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance. Proc Natl Acad Sci USA 102: 15167-72. Bi, X., S.C. Wei, and Y.S. Rong. 2004. Telomere protection without a telomerase; the role of

9. Blackburn, E.H. 1992. Telomerases. Annu Rev Biochem 61: 113-29.

10. Blumenstiel, J.P. and D.L. Hartl. 2005. Evidence for maternally transmitted small interfering RNA in the repression of transposition in Drosophila virilis. Proc Natl Acad Sci USA 102: 15965-70.

11. Bohnsack, M.T., K. Czaplinski, and D. Gorlich. 2004. Exportin 5 is a RanGTP-dependentdsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10: 185-91.

12. Boivin, A., C. Gaily, S. Netter, D. Anxolabehere, and S. Ronsseray. 2003. Telomeric associated sequences of Drosophila recruit polycomb-group proteins in vivo and can induce pairing-sensitive repression. Genetics 164: 195-208.

13. Brennecke, J., A.A. Aravin, A. Stark, M. Dus, M. Kellis, R. Sachidanandam, and G.J. Hannon. 2007. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128: 1089-103.

14. Bryan, T.M. and T.R. Cech. 1999. Telomerase and the maintenance of chromosome ends. Curr Opin Cell Biol 11: 318-24.

15. Carrington, J.C. and V. Ambros. 2003. Role of microRNAs in plant and animal development. Science 301: 336-8.

16. Casacuberta, E. and M.L. Pardue. 2002. Coevolution of the telomeric retrotransposons across Drosophila species. Genetics 161: 1113-24.

17. Casacuberta, E. and M.L. Pardue. 2003a. HeT-A elements in Drosophila virilis: retrotransposon telomeres are conserved across the Drosophila genus. Proc Natl Acad Sci USA 100: 14091-6.

18. Casacuberta, E. and M.L. Pardue. 2003b. Transposon telomeres are widely distributed in the Drosophila genus: TART elements in the virilis group. Proc Natl Acad Sci U SA 100: 3363-8.

19. Cenci, G., G. Siriaco, G.D. Raffa, R. Kellum, and M. Gatti. 2003. The Drosophila HOAP protein is required for telomere capping. Nat Cell Biol 5: 82-4.

20. Cenci, G., L. Ciapponi, and M. Gatti. 2005. The mechanism of telomere protection: a comparison between Drosophila and humans. Chromosoma 114: 135-45.

21. Chan, S.W. and E.H. Blackburn. 2002. New ways not to make ends meet: telomerase, DNA damage proteins and heterochromatin. Oncogene 21: 553-63.

22. Chapon, C., T.R. Cech, and A.J. Zaug. 1997. Polyadenylation of telomerase RNA in budding yeast. RNA 3: 1337-51.

23. Ciapponi, L., G. Cenci, J. Ducau, C. Flores, D. Johnson-Schlitz, M.M. Gorski, W.R. Engels, and M. Gatti. 2004. The Drosophila Mrel 1/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage. Curr Biol 14: 1360-6.

24. Collins, R.E. and X. Cheng. 2005. Structural domains in RNAi. FEBS Lett 579: 5841-9.

25. Cox, D.N., A. Chao, J. Baker, L. Chang, D. Qiao, and H. Lin. 1998. A novel class ofevolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev 12: 3715-27.

26. Cox, D.N., A. Chao, and H. Lin. 2000. piwi encodes a nucleoplasmic factor whose activitymodulates the number and division rate of germline stem cells. Development 127: 50314.

27. Czech, B., C.D. Malone, R. Zhou, A. Stark, C. Schlingeheyde, M. Dus, N. Perrimon, M. Kellis, J.A. Wohlschlegel, R. Sachidanandam, G.J. Hannon, and J. Brennecke. 2008. An endogenous small interfering RNA pathway in Drosophila. Nature 453: 798-802.

28. Danilevskaya, O.N., I.R. Arkhipova, K.L. Traverse, and M.L. Pardue. 1997. Promoting intandem: the promoter for telomere transposon HeT-A and implications for the evolution of retroviral LTRs. Cell 88: 647-55.

29. Danilevskaya, O.N., K.L. Traverse, N.C. Hogan, P.G. DeBaryshe, and M.L. Pardue. 1999. The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol Cell Biol 19: 873-81.

30. David, A., N. Mabjeesh, I. Azar, S. Biton, S. Engel, J. Bernstein, J. Romano, Y. Avidor, T.

31. Day, A., M. Schirmer-Rahire, M.R. Kuchka, S.P. Mayfield, and J.D. Rochaix. 1988. Atransposon with an unusual arrangement of long terminal repeats in the green alga Chlamydomonas reinhardtii. EmboJl: 1917-27.

32. Denli, A.M., B.B. Tops, R.H. Plasterk, R.F. Ketting, and G.J. Hannon. 2004. Processing of primary microRNAs by the Microprocessor complex. Nature 432: 231-5.

33. Desset, S., C. Meignin, B. Dastugue, and C. Vaury. 2003. COM, a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster. Genetics 164: 501-9.

34. Di Nocera, P.P. and I.B. Dawid. 1983. Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci USA 80: 7095-8.

35. Diede, S.J. and D.E. Gottschling. 1999. Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell 99: 723-33.

36. Fanti, L., D.R. Dorer, M. Berloco, S. Henikoff, and S. Pimpinelli. 1998a. Heterochromatin protein 1 binds transgene arrays. Chromosoma 107: 286-92.

37. Fanti, L., G. Giovinazzo, M. Berloco, and S. Pimpinelli. 1998b. The heterochromatin protein 1 prevents telomere fusions in Drosophila. Mol Cell 2: 527-38.

38. George, J. A. and M.L. Pardue. 2003. The promoter of the heterochromatic Drosophila telomeric retrotransposon, HeT-A, is active when moved into euchromatic locations. Genetics 163: 625-35.

39. George, J.A., P.G. DeBaryshe, K.L. Traverse, S.E. Celniker, and M.L. Pardue. 2006. Genomic organization of the Drosophila telomere retrotransposable elements. Genome Res 16: 1231-40.

40. Ghildiyal, M., H. Seitz, M.D. Horwich, C. Li, T. Du, S. Lee, J. Xu, E.L. Kittler, M.L. Zapp, Z. Weng, and P.D. Zamore. 2008. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320: 1077-81.

41. Gillespie, D.E. and C.A. Berg. 1995. Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases. Genes Dev 9: 2495-508.

42. Girard, A., R. Sachidanandam, G.J. Hannon, and M.A. Carmell. 2006. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442: 199-202.

43. Golubovsky, M.D., A.Y. Konev, M.F. Walter, H. Biessmann, and J.M. Mason. 2001. Terminal retrotransposons activate a subtelomeric white transgene at the 2L telomere in Drosophila. Genetics 158: 1111-23.

44. Gregory, R.I., K.P. Yan, G. Amuthan, T. Chendrimada, B. Doratotaj, N. Cooch, and R.

45. Shiekhattar. 2004. The Microprocessor complex mediates the genesis of microRNAs. Nature 432: 235-40.

46. Greider, C.W. and E.H. Blackburn. 1985. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43: 405-13.

47. Griffith, J.D., L. Comeau, S. Rosenfield, R.M. Stansel, A. Bianchi, H. Moss, and T. de Lange. 1999. Mammalian telomeres end in a large duplex loop. Cell 97: 503-14.

48. Gunawardane, L.S., K. Saito, K.M. Nishida, K. Miyoshi, Y. Kawamura, T. Nagami, H. Siomi, and M.C. Siomi. 2007. A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila. Science 315: 1587-90.

49. Haley, B. and P.D. Zamore. 2004. Kinetic analysis of the RNAi enzyme complex. Nat Struct Mol Biol 11: 599-606.

50. Hall, I.M., K. Noma, and S.I. Grewal. 2003. RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast. Proc Natl Acad Sci USA 100: 1938.

51. Hall, I.M., G.D. Shankaranarayana, K. Noma, N. Ayoub, A. Cohen, and S.I. Grewal. 2002. Establishment and maintenance of a heterochromatin domain. Science 297: 2232-7.

52. Hamilton, A., O. Voinnet, L. Chappell, and D. Baulcombe. 2002. Two classes of short interfering RNA in RNA silencing. Embo J21: 4671-9.

53. Han, J., Y. Lee, K.H. Yeom, Y.K. Kim, H. Jin, and V.N. Kim. 2004. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18: 3016-27.

54. Han, J., Y. Lee, K.H. Yeom, J.W. Nam, I. Heo, J.K. Rhee, S.Y. Sohn, Y. Cho, B.T. Zhang, and V.N. Kim. 2006. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125: 887-901.

55. Harris, A.N. and P.M. Macdonald. 2001. Aubergine encodes a Drosophila polar granulecomponent required for pole cell formation and related to eIF2C. Development 128: 2823-32.

56. Henikoff, S. 1997. Nuclear organization and gene expression: homologous pairing and longrange interactions. Curr Opin Cell Biol 9: 388-95.

57. Henson, J.D., A.A. Neumann, T.R. Yeager, and R.R. Reddel. 2002. Alternative lengthening of telomeres in mammalian cells. Oncogene 21: 598-610.

58. Herr, A.J., M.B. Jensen, T. Dalmay, and D.C. Baulcombe. 2005. RNA polymerase IV directs silencing of endogenous DNA. Science 308: 118-20.

59. Horwich, M.D., C. Li, C. Matranga, V. Vagin, G. Farley, P. Wang, and P.D. Zamore. 2007. The Drosophila RNA methyltransferase, DmHenl, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr Biol 17: 1265-72.

60. Hsu, H.L., D. Gilley, S.A. Galande, M.P. Hande, B. Allen, S.H. Kim, G.C. Li, J. Campisi, T. Kohwi-Shigematsu, and D.J. Chen. 2000. Ku acts in a unique way at the mammalian telomere to prevent end joining. Genes Dev 14: 2807-12.

61. Humphreys, D.T., B.J. Westman, D.I. Martin, and T. Preiss. 2005. MicroRNAs controltranslation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc Natl Acad Sci USA 102: 16961-6.

62. Kahn, T., M. Savitsky, and P. Georgiev. 2000. Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms. Mol Cell Biol 20:7634-42.

63. Kalmykova, A.I., M.S. Klenov, and Y.A. Gvozdev. 2005. Argonaute protein PIWI controlsmobilization of retrotransposons in the Drosophila male germline. Nucleic Acids Res 33: 2052-9.

64. Kanoh, J., M. Sadaie, T. Urano, and F. Ishikawa. 2005. Telomere binding protein Tazlestablishes Swi6 heterochromatin independently of RNAi at telomeres. Curr Biol 15: 1808-19.

65. Kawamura, Y., K. Saito, T. Kin, Y. Ono, K. Asai, T. Sunohara, T.N. Okada, M.C. Siomi, and H. Siomi. 2008. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453: 793-7.

66. Kelleher, C., M.T. Teixeira, K. Forstemann, and J. Lingner. 2002. Telomerase: biochemical considerations for enzyme and substrate. Trends Biochem Sci 27: 572-9.

67. Kim, N.W., M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, and J.W. Shay. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266: 2011-5.

68. Kim, Y.N. 2005. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6: 376-85.

69. Kim, Y.K. and V.N. Kim. 2007. Processing of intronic microRNAs. Embo J 26: 775-83.

70. Kishi, S., G. Wulf, M. Nakamura, and K.P. Lu. 2001. Telomeric protein Pin2/TRF1 inducesmitotic entry and apoptosis in cells with short telomeres and is down-regulated in human breast tumors. Oncogene 20: 1497-508.

71. Matranga, C., Y. Tomari, C. Shin, D.P. Bartel, and P.D. Zamore. 2005. Passenger-strandcleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123: 607-20.

72. Matzke, M.A. and J.A. Birchler. 2005. RNAi-mediated pathways in the nucleus. Nat Rev Genet 6: 24-35.

73. Maxwell, P.H., J.M. Belote, and R.W. Levis. 2006. Identification of multiple transcriptioninitiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons. Nucleic Acids Res 34: 5498-507.

74. Megosh, H.B., D.N. Cox, C. Campbell, and H. Lin. 2006. The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr Biol 16: 1884-94.

75. Meister, G., M. Landthaler, A. Patkaniowska, Y. Dorsett, G. Teng, and T. Tuschl. 2004. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15: 18597.

76. Melnikova, L., H. Biessmann, and P. Georgiev. 2005. The Ku protein complex is involved in length regulation of Drosophila telomeres. Genetics 170: 221-35.

77. Mikhailovsky, S. T. Belenkaya, and P. Georgiev. 1999. Broken chromosomal ends can be elongated by conversion in Drosophila melanogaster. Chromosoma 108: 114-20.

78. Miyoshi, K., H. Tsukumo, T. Nagami, H. Siomi, and M.C. Siomi. 2005. Slicer function of

79. Drosophila Argonautes and its involvement in RISC formation. Genes Dev 19: 2837-48.

80. Mochizuki, K. and M.A. Gorovsky. 2005. A Dicer-like protein in Tetrahymena has distinct functions in genome rearrangement, chromosome segregation, and meiotic prophase. Genes Dev 19: 77-89.

81. Mourelatos, Z., J. Dostie, S. Paushkin, A. Sharma, B. Charroux, L. Abel, J. Rappsilber, M.

82. Mann, and G. Dreyfuss. 2002. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16: 720-8.

83. Noma, K., T. Sugiyama, H. Cam, A. Verdel, M. Zofall, S. Jia, D. Moazed, and S.I. Grewal.2004. RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nat Genet 36: 1174-80.

84. Nussenzweig, A., C. Chen, V. da Costa Soares, M. Sanchez, K. Sokol, M.C. Nussenzweig, and G.C. Li. 1996. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382: 551-5.

85. O'Connor, M.S., A. Safari, D. Liu, J. Qin, and Z. Songyang. 2004. The human Rapl protein complex and modulation of telomere length. J Biol Chem 279: 28585-91.

86. Oikemus, S.R., N. McGinnis, J. Queiroz-Machado, H. Tukachinsky, S. Takada, C.E. Sunkel, and M.H. Brodsky. 2004. Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect. Genes Dev 18: 1850-61.

87. Okamura, K., S. Balla, R. Martin, N. Liu, and E.C. Lai. 2008. Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat Struct Mol Biol 15: 581-90.

88. Onodera, Y., J.R. Haag, T. Ream, P.C. Nunes, O. Pontes, and C.S. Pikaard. 2005. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120: 613-22.

89. Pal-Bhadra, M., B.A. Leibovitch, S.G. Gandhi, M. Rao, U. Bhadra, J.A. Birchler, and S.C. Elgin. 2004. Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303: 669-72.

90. Palm, W. and T. de Lange. 2008. How Shelterin Protects Mammalian Telomeres. Annu Rev Genet.

91. Pane, A., K. Wehr, and T. Schupbach. 2007. zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline. Dev Cell 12: 851-62.

92. Pardue, M.L., O.N. Danilevskaya, K. Lowenhaupt, J. Wong, and K. Erby. 1996. The gag coding region of the Drosophila telomeric retrotransposon, HeT-A, has an internal frame shift and a length polymorphic region. JMol Evol 43: 572-83.

93. Pardue, M.L. and P.G. DeBaryshe. 2003. Retrotransposons provide an evolutionarily robust non-telomerase mechanism to maintain telomeres. Annu Rev Genet 37: 485-511.

94. Pelisson, A., S.U. Song, N. Prud'homme, P.A. Smith, A. Bucheton, and V.G. Corces. 1994.

95. Gypsy transposition correlates with the production of a retroviral envelope-like protein under the tissue-specific control of the Drosophila flamenco gene. Embo J13: 4401-11.

96. Peterson, S.E., A.E. Stellwagen, S.J. Diede, M.S. Singer, Z.W. Haimberger, C.O. Johnson, M. Tzoneva, and D.E. Gottschling. 2001. The function of a stem-loop in telomerase RNA is linked to the DNA repair protein Ku. Nat Genet 27: 64-7.

97. Plasterk, R.H. 2006. Micro RNAs in animal development. Cell 124: 877-81.

98. Prescott, J. and E.H. Blackburn. 1997. Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro. Genes Dev 11: 528-40.

99. Rand, T.A., S. Petersen, F. Du, and X. Wang. 2005. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123: 621-9.

100. Rashkova, S., S.E. Karam, R. Kellum, and M.L. Pardue. 2002a. Gag proteins of the two

101. Drosophila telomeric retrotransposons are targeted to chromosome ends. J Cell Biol 159: 397-402.

102. Rashkova, S., S.E. Karam, and M.L. Pardue. 2002b. Element-specific localization of Drosophila retrotransposon Gag proteins occurs in both nucleus and cytoplasm. Proc Natl Acad Sci USA 99: 3621-6.

103. Rashkova, S., A. Athanasiadis, and M.L. Pardue. 2003. Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons. J Virol 77: 6376-84.

104. Robert, V., N. Prud'homme, A. Kim, A. Bucheton, and A. Pelisson. 2001. Characterization of the flamenco region of the Drosophila melanogaster genome. Genetics 158: 701-13.

105. Romero, D.P. and E.H. Blackburn. 1991. A conserved secondary structure for telomerase RNA. Cell 67: 343-53.

106. Saito, K., K.M. Nishida, T. Mori, Y. Kawamura, K. Miyoshi, T. Nagami, H. Siomi, and M.C. Siomi. 2006. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 20: 2214-22.

107. Sambrook and Russel. 2001. Molecular cloning. New York, Cold Spring Harbor press.

108. Samper, E., F.A. Goytisolo, P. Slijepcevic, P.P. van Buul, and M.A. Blasco. 2000. Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang. EMBO Rep 1: 244-52.

109. Sarot, E., G. Payen-Groschene, A. Bucheton, and A. Pelisson. 2004. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166: 1313-21.

110. Savitsky, M., O. Kravchuk, L. Melnikova, and P. Georgiev. 2002. Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster. Mol Cell Biol 22: 3204-18.

111. Savitsky, M., D. Kwon, P. Georgiev, A. Kalmykova, and V. Gvozdev. 2006. Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline. Genes Dev 20: 345-54.

112. Seto, A.G., A.J. Livengood, Y. Tzfati, E.H. Blackburn, and T.R. Cech. 2002. A bulged stem tethers Estlp to telomerase RNA in budding yeast. Genes Dev 16: 2800-12.

113. Seto, A.G., A.J. Zaug, S.G. Sobel, S.L. Wolin, and T.R. Cech. 1999. Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle. Nature 401: 177-80.

114. Sheen, F.M. and R.W. Levis. 1994. Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. Proc Natl Acad Sci U SA 91: 12510-4.

115. Shi, H., A. Djikeng, C. Tschudi, and E. Ullu. 2004. Argonaute protein in the early divergent eukaryote Trypanosoma brucei: control of small interfering RNA accumulation and retroposon transcript abundance. Mol Cell Biol 24: 420-7.

116. Sijen, T. and R.H. Plasterk. 2003. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature 426: 310-4.

117. Silva, E., S. Tiong, M. Pedersen, E. Homola, A. Royou, B. Fasulo, G. Siriaco, and S.D.

118. Campbell. 2004. ATM is required for telomere maintenance and chromosome stability during Drosophila development. Curr Biol 14: 1341-7.

119. Siolas, D., C. Lerner, J. Burchard, W. Ge, P.S. Linsley, P.J. Paddison, G.J. Hannon, and M.A. Cleary. 2005. Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23: 227-31.

120. Siriaco, G.M., G. Ccnci, A. Haoudi, L.E. Champion, C. Zhou, M. Gatti, and J.M. Mason. 2002. Telomere elongation (Tel), a new mutation in Drosophila melanogaster that produces long telomeres. Genetics 160: 235-45.

121. Smith, S., I. Giriat, A. Schmitt, and T. de Lange. 1998. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282: 1484-7.

122. Song, K., D. Jung, Y. Jung, S.G. Lee, and I. Lee. 2000. Interaction of human Ku70 with TRF2. FEBS Lett 481: 81-5.

123. Song, Y.H., G. Mirey, M. Betson, D.A. Haber, and J. Settleman. 2004. The Drosophila ATM ortholog, dATM, mediates the response to ionizing radiation and to spontaneous DNA damage during development. Curr Biol 14: 1354-9.

124. Sowd, G., M. Lei, and P.L. Opresko. 2008. Mechanism and substrate specificity of telomericprotein POT1 stimulation of the Werner syndrome helicase. Nucleic Acids Res 36: 424256.

125. Stapleton, W., S. Das, and B.D. McKee. 2001. A role of the Drosophila homeless gene in repression of Stellate in male meiosis. Chromosoma 110: 228-40.

126. Stellwagen, A.E., Z.W. Haimberger, J.R. Veatch, and D.E. Gottschling. 2003. Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev 17: 2384-95.

127. Sugiyama, T., H. Cam, A. Verdel, D. Moazed, and S.I. Grewal. 2005. RNA-dependent RNApolymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production. Proc Natl Acad Sci USA 102: 152-7.

128. Sugiyama, T., H.P. Cam, R. Sugiyama, K. Noma, M. Zofall, R. Kobayashi, and S.I. Grewal. 2007. SHREC, an effector complex for heterochromatic transcriptional silencing. Cell 128:491-504.

129. Svoboda, P., P. Stein, M. Anger, E. Bernstein, G.J. Hannon, and R.M. Schultz. 2004. RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev Biol 269:276-85.

130. Tabara, H., M. Sarkissian, W.G. Kelly, J. Fleenor, A. Grishok, L. Timmons, A. Fire, and C.C. Mello. 1999. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99: 123-32.

131. Taddei, A., F. Hediger, F.R. Neumann, and S.M. Gasser. 2004. The function of nuclear architecture: a genetic approach. Annu Rev Genet 38: 305-45.

132. Taggart, A.K. and V.A. Zakian. 2003. Telomerase: what are the Est proteins doing? Curr Opin Cell Biol 15:275-80.

133. Tomari, Y., C. Matranga, B. Haley, N. Martinez, and P.D. Zamore. 2004. A protein sensor for siRNA asymmetry. Science 306: 1377-80.

134. Torok, T., C. Benitez, S. Takacs, and H. Biessmann. 2007. The protein encoded by the gene proliferation disrupter (prod) is associated with the telomeric retrotransposon array in Drosophila melanogaster. Chromosoma 116: 185-95.

135. Vagin, V.V., A. Sigova, C. Li, H. Seitz, V. Gvozdev, and P.D. Zamore. 2006. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313: 320-4.

136. Villasante, A., J.P. Abad, R. Planello, M. Mendez-Lago, S.E. Celniker, and B. de Pablos. 2007. Drosophila telomeric retrotransposons derived from an ancestral element that was recruited to replace telomerase. Genome Res 17: 1909-18.

137. Volpe, T.A., C. Kidner, I.M. Hall, G. Teng, S.I. Grewal, and R.A. Martienssen. 2002. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297: 1833-7.

138. Walter, M.F. and H. Biessmann. 2004. Expression of the telomeric retrotransposon HeT-A in

139. Drosophila melanogaster is correlated with cell proliferation. Dev Genes Evol 214: 211-9.

140. Wienholds, E. and R.H. Plasterk. 2005. MicroRNA function in animal development. FEBS Lett 579:5911-22.

141. Wu-Scharf, D., B. Jeong, C. Zhang, and H. Cerutti. 2000. Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Science 290: 1159-62.

142. Yi, R., Y. Qin, I.G. Macara, and B.R. Cullen. 2003. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17: 3011-6.

143. Zappulla, D.C. and T.R. Cech. 2004. Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc Natl Acad Sci USA 101: 10024-9.

144. Zijlmans, J.M., U.M. Martens, S.S. Poon, A.K. Raap, H.J. Tanke, R.K. Ward, and P.M.1.nsdorp. 1997. Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc Natl Acad Sci USA 94: 7423-8.

145. Zilberman, D., X. Cao, L.K. Johansen, Z. Xie, J.C. Carrington, and S.E. Jacobsen. 2004. Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr Biol 14: 1214-20.1. Благодарности