1. RNA silencing Jennifer Grier 1 November 2012

2. Overview Timing Mechanisms Long non-coding RNA – Xist – Air – HOTAIR Short non-coding RNA – siRNA – miRNA – piRNA

3. Timing/Location of RNA silencing Nucleus CDGS: chromatin-dependent gene silencing – TGS: Transcriptional gene silencing – CTGS: Co-transcriptional gene silencing Cytoplasm PTGS: post-transcriptional gene silencing

4. Mechanisms of RNA silencing Heterochromatin Formation – TGS, CTGS Enhancer - repression of silencing Transcript degradation – PTGS Translational arrest – PTGS

5. Mechanisms of RNA silencing Heterochromatin Formation – TGS, CTGS Adapted from: Kevin V. Morris. Oligonucleotides. 2009 December;19(4):299-305.

6. Mechanisms of RNA silencing Heterochromatin Formation – TGS, CTGS Enhancer - repression of silencing X X X Adapted from: Kevin V. Morris. Oligonucleotides. 2009 December;19(4):299-305.

7. Mechanisms of RNA silencing Transcript degradation – PTGS Translational arrest – PTGS Moazed, D. Nature. 2009 Jan 22;457(7228):413-20.Nature. (arrest)

8. Long non-coding RNA’s Abbreviated as lncRNA, or lincRNA (long intergenic non-coding RNA) Defined as: – >200 bp in length (can be up to 100 kb) – Not processed – Non-protein coding Very prevalent in genome

9. Mercer et al., NRG 2009 Found in many places in the genome lncRNA Gene transcript

10. How do they work? chromatin regulator recruitment Mercer et al., NRG 2009

11. How do they work? chromatin regulator recruitment RNA binding protein recruitment RNA binding protein inhibits HATs Mercer et al., NRG 2009

12. How do they work? chromatin regulator recruitment RNA binding protein recruitment TF recruitment/nuclear import Mercer et al., NRG 2009 Enhancer RNA Some lncRNA in HOX cluster interact with Trithorx resulting in H3K4me3

13. How do they work? chromatin regulator recruitment RNA binding protein recruitment TF recruitment (triple helix) interference with binding or activity of the general transcriptional machinery Mercer et al., NRG 2009

14. Koziol and Rinn COGD, 2010 Mechanisms of Chromatin Regulation Important to note: Cis or Trans

15. Mechanisms of Chromatin Regulation Koziol and Rinn COGD, 2010 Tethers: sequence specificity

16. Mechanisms of Chromatin Regulation Koziol and Rinn COGD, 2010 Tethers: sequence specificity Acts as Scaffold

17. Mechanisms of Chromatin Regulation Koziol and Rinn COGD, 2010 Tethers: sequence specificity Acts as Scaffold Regulates activity

18. Mechanisms of Chromatin Regulation Koziol and Rinn COGD, 2010 Tethers: sequence specificity Acts as Scaffold Regulates activity Mediates long range interactions

19. Mechanisms of Chromatin Regulation Tethers: sequence specificity Acts as Scaffold Regulates activity Mediates long range interactions Means of carrying epigenetic information from mother to daughter cell

20. X inactivation Pontier, DB and Gribnau, J. Hum Genet. 2011 August; 130(2): 223–236. FISH = fluorescent in situ hybridization polycomb

21. Magenta: Jpx RNA, green XIST Tian et al. Cell 2010 X inactivation: additional players X-inactivation center 4 ncRNAs Xi : Xist and RepA – RepA binds PRC2 Xa: Tsix cis-Xist repressor Both: Jpx cis- and trans-Xist activator

22. Xist only expressed in heterochromatin RepA stem loop binds PRC2 – leading to H3K27me3 in cis on Xi Allows activation by JPX Caley et al., The Scientific World Journal 2010 X inactivation: additional players Tsix recruits Dnmt3a – Methylates Xist promoter Activation by JPX is blocked

23. Ideraabdullah, Mut. Res., 2008 AIR recruits G9a (HMT) Results in H3K9me at imprinted gene promoters in cis Igf2r/Air - lncRNA mediated imprinting

24. HOTAIR Gupta et al. Nature 2101 Expressed from HOXC locus Represses in trans Developmentally regulated (Hox genes)

25. Tsai et al., Science 2010 HOTAIR Binds: – PRC2 – EZH2 (HMT) – CoREST (HDAC) – LSD1 (H3K4me demethlase)

26. Moazed Nature 2009 (RNAi) Small non-coding RNAs

27. RNAi = RNA-interference Double stranded (ds) RNA induces homology-dependent degradation of cognate RNA and depletion of protein over time

28. Andrew Z. Fire Craig C. Mello 1/2 of the prize USA Stanford University University of Massachusetts School of MedicineMedical School Stanford, CA, USA Worcester, MA, USA The Nobel Prize in Physiology or Medicine 2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA"

29. Functions: viral silencing in plants suppression of transposable elements silencing of repetitive sequences heterochromatin formation transgene silencing Small interfering RNAs: siRNAs

30. Source for siRNA: Endogenous Exogenous

31. How are small ncRNA generated? siRNA – natural cis antisense siRNAs – repeat associated siRNA Result in dsRNA products PolII/V transcribed or bi-directional transcription Moazed Nature 2009

32. How are small ncRNA generated? From aberrant transcripts By RdRp: RNA-dependent RNA polymerase

33. How are small ncRNA generated? miRNA – from miRNA genes (non-coding) – found within lncRNAs and coding genes – PolII transcribed – Forms a hairpin

34. Tamari and Zamore Prespectives: machines for RNAi Genes &Dev.19:517-529 (2005) Processing of small ncRNAs

35. Processing of miRNAs

36. DCL Arabidopsis initially isolated as developmental mutant! Dicer Ribonuclease III homolog; helicase Drosophila, C.elegans, mouse, fungi Role for a bidentate ribonuclease in the initiation step of RNA interference Emily Bernstein, Amy A. Caudy, Scott M. Hammond & Gregory J. Hannon NATURE | VOL 409 | 18 JANUARY 2001 Processing of miRNAs

37. Binds precursor ds or miRNA through PAZ domain Cleaves precursor through ribonuclease III domain Spacing between PAZ and RIII domains determine size and cut location resulting in staggered cuts Moazed Nature 2009 Processing of miRNAs

38. Functions of small ncRNA

39. Argonaute family of proteins – bind miRNA or siRNA or piRNA At least two classes – AGO-like – PIWI-like Important Components

40. AGO (Argonaute) PIWI domain binds 5’ end small RNA (RNAse H-like fold) PAZ domain binds 3’ end small RNA guide strand Mid domain binds CAP Slicer activity (some AGOs) required for siRNA, most plant miRNA makes a cut in target RNA leading to degradation Catalytically inactive AGOs lead to inhibition of translation (stalling) Multi turnover enzyme

41. RNA-directed RNA polymerase RdRP: Amplification Transport (systemic RNAi) Heterochromatin formation Not required for Drosophila or mammalian RNAi Important Components Moazed Nature 2009

42. RITS complex RNA induced transcriptional silencing Ago1 (Argonaute, binds siRNAs) Chp1 (chromodomain, binds H3K9me) Tas3 (binds Ago1 and Chp1, spreading) siRNAs (small inhibitory RNA) Like RISC: effector complex, bind small RNA Important Components

43. Transcriptional Gene Silencing Djupedal and Ekwall, Cell Research, 2009

44. Transcriptional Gene Silencing Djupedal and Ekwall, Cell Research, 2009

45. Transcriptional Gene Silencing Djupedal and Ekwall, Cell Research, 2009

46. initiation amplification de novo DNA methylation Simon and Meyers COPB 2011 RNA induced DNA Methylation -- Role of siRNAs in ESTABLISHMENT of transcriptional gene -- silencing first discovered in plants RNA methods of TGS

47. tasiRNAs Djupedal and Ekwall, Cell Research, 2009, V PolIV and V in TGS

48. PolIV PolV Wierzbicki et al. Cell, 2008

49. PolIV and V in TGS

50. Wierzbicki et al. Cell, 2008 PolIV and V in TGS

51. Haag and Pikaard, Nat. Rev. MCB, 2011 RNA methods of TGS (in plants)

52. Maintenance spreading RNA methods of TGS (in plants)

53. TGS speading A. thaliana RNA methods of TGS (in plants)

54. Khraiwesh et al., Cell 2010 High levels of miRNA: Cause miRNA:mRNA duplex formation Trigger DNA methylation (for example in response to hormone treatment) Also described in mammals Kim et al, PNAS 2008 In moss: role of miRNA in DNA methylation RNA methods of TGS (in plants)

55. Role of siRNAs in silencing (plants) triggers DNA methylation recruitment of H3K9me role in maintenance of DNA methylation spreading

56. Lejeune and Allshire, COCB 2011 RNA methods of TGS (S. pombe)

57. Role of siRNAs in silencing (S. pombe) initiation of all heterochromatin together with Clr4 (HMT)! maintentance of centromeric heterochromatin tethered via RITS, Clr4-dependent RITS brings in nascent transcript RNA methods of TGS (S. pombe)

58. Potential for Inheritance? 1.siRNAs inherited, trigger H3K9me 2.Positive feedback between siRNAs and H3K9me: amplification and stabilization YES!

59. piRNA Found in animals Role in germline Silencing of of repetitive DNA transposons, subtelomeric regions pericentromeric regions PROTECTING THE GERMLINE

60. PIWI: role in piRNA generation Moazed Nature 2009 PROTECTING THE GERMLINE

61. piRNA 2ndary piRNA Dicer independent PIWI-AGO example of AGO activity

62. PIWI associates with HP1 D.m. piRNAs from follicle cells into oocytes also linked to triggering DNA methylation Accumulate at time of erasure and re-establishment of DNA methylation PROTECTING THE GERMLINE

63. Slotkin et al., Cell 2009 Similar observations for female gametophyte PROTECTING THE GERMLINE

64. In mammals: TE silencing via DNA methylation pericentromeric RNA involved Occurs early after fertilization Satenard et al. Nat. Cell. Biol. 2010 Probst et al. Dev Cell 2010 PROTECTING THE GERMLINE

65. AGO-linked activities Transcriptional Gene Silencing

66. PolIV PolII TGS PTGS (post transcriptional gene silencing) message cleavage inhibition of translation (small and large ncRNAs)

67. PTGS siRNA

68. PTGS RISC complex RNA-induced silencing complex si RNA – AGO – DCR – dsRNA binding protein (TRBP) miRNA – AGO – GW182

69. FEBS letters 2005 PTGS

70. Small ncRNA movement some miRNAs can move (short distance) siRNAs can move: systemic responses Systemic Silencing

71. Summary types of long noncoding RNAs definition, where arise roles of lnc RNAs in chromatin regulation types of small noncoding RNAs definition, biogenesis roles in chromatin regulation initiation and maintenance Potential for epigenetic inheritance Role in germline Systemic silencing: why important?