RNA silencing

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 X • Enhancer - repression of silencing X X Adapted from: Kevin V. Morris. Oligonucleotides. 2009 December;19(4):299-305.

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

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

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 Enhancer RNA Some lncRNA in HOX cluster interact with Trithorx resulting in H3K4me3 Mercer et al., NRG 2009

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

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

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

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

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

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

21. 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 Magenta: Jpx RNA, green XIST Tian et al. Cell 2010

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

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

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

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

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

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

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

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

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 • ByRdRp: RNA-dependent RNA polymerase Moazed Nature 2009

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

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

35. Processing of miRNAs

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

37. Processing of miRNAs 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

38. Functions of small ncRNA

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

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

42. Important Components 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

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

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

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

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

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