RNA silencing Jennifer Grier 1 November 2012 Overview • Timing • Mechanisms • Long non-coding RNA – Xist – Air – HOTAIR • Short non-coding RNA – siRNA – miRNA – piRNA 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 Mechanisms of RNA silencing • Heterochromatin Formation – TGS, CTGS • Enhancer - repression of silencing • Transcript degradation – PTGS • Translational arrest – PTGS Mechanisms of RNA silencing • Heterochromatin Formation – TGS, CTGS Adapted from: Kevin V. Morris. Oligonucleotides. 2009 December;19(4):299-305. 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. Mechanisms of RNA silencing (arrest) • Transcript degradation – PTGS • Translational arrest – PTGS Moazed, D. Nature. 2009 Jan 22;457(7228):413-20. 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 Found in many places in the genome lncRNA Gene transcript Mercer et al., NRG 2009 How do they work? • chromatin regulator recruitment Mercer et al., NRG 2009 How do they work? • chromatin regulator recruitment • RNA binding protein recruitment RNA binding protein inhibits HATs Mercer et al., NRG 2009 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 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 Mechanisms of Chromatin Regulation Important to note: Cis or Trans Koziol and Rinn COGD, 2010 Mechanisms of Chromatin Regulation • Tethers: sequence specificity Koziol and Rinn COGD, 2010 Mechanisms of Chromatin Regulation • Tethers: sequence specificity • Acts as Scaffold Koziol and Rinn COGD, 2010 Mechanisms of Chromatin Regulation • Tethers: sequence specificity • Acts as Scaffold • Regulates activity Koziol and Rinn COGD, 2010 Mechanisms of Chromatin Regulation • Tethers: sequence specificity • Acts as Scaffold • Regulates activity • Mediates long range interactions Koziol and Rinn COGD, 2010 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 X inactivation polycomb FISH = fluorescent in situ hybridization Pontier, DB and Gribnau, J. Hum Genet. 2011 August; 130(2): 223–236. X inactivation: additional players X-inactivation center 4 ncRNAs • Xi : Xist and RepA – RepA binds PRC2 Magenta: Jpx RNA, green XIST • Xa: Tsix • cis-Xist repressor • Both: Jpx • cis- and trans-Xist activator Tian et al. Cell 2010 X inactivation: additional players Tsix recruits Dnmt3a – Methylates Xist promoter Activation by JPX is blocked 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 Igf2r/Air - lncRNA mediated imprinting AIR recruits G9a (HMT) Results in H3K9me at imprinted gene promoters in cis Ideraabdullah, Mut. Res., 2008 HOTAIR • Expressed from HOXC locus • Represses in trans Developmentally regulated (Hox genes) Gupta et al. Nature 2101 HOTAIR • Binds: – PRC2 – EZH2 (HMT) – CoREST (HDAC) – LSD1 (H3K4me demethlase) Tsai et al., Science 2010 Small non-coding RNAs (RNAi) Moazed Nature 2009 RNAi = RNA-interference Double stranded (ds) RNA induces homology-dependent degradation of cognate RNA and depletion of protein over time The Nobel Prize in Physiology or Medicine 2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA" Andrew Z. Fire 1/2 of the prize USA Stanford University School of Medicine Stanford, CA, USA Craig C. Mello 1/2 of the prize USA University of Massachusetts Medical School Worcester, MA, USA Small interfering RNAs: siRNAs Functions: viral silencing in plants suppression of transposable elements silencing of repetitive sequences heterochromatin formation transgene silencing Source for siRNA: Endogenous Exogenous 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 How are small ncRNA generated? • From aberrant transcripts • By RdRp: RNA-dependent RNA polymerase Moazed Nature 2009 How are small ncRNA generated? • miRNA – from miRNA genes (non-coding) – found within lncRNAs and coding genes – PolII transcribed – Forms a hairpin Processing of small ncRNAs Tamari and Zamore Prespectives: machines for RNAi Genes &Dev.19:517-529 (2005) Processing of miRNAs Processing of miRNAs 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 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 Functions of small ncRNA Important Components • Argonaute family of proteins – bind miRNA or siRNA or piRNA • At least two classes – AGO-like – PIWI-like 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 Important Components RNA-directed RNA polymerase RdRP: Amplification Transport (systemic RNAi) Heterochromatin formation Not required for Drosophila or mammalian RNAi Moazed Nature 2009 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 Transcriptional Gene Silencing Djupedal and Ekwall, Cell Research, 2009 Transcriptional Gene Silencing Djupedal and Ekwall, Cell Research, 2009 Transcriptional Gene Silencing Djupedal and Ekwall, Cell Research, 2009 RNA methods of TGS RNA induced DNA Methylation initiation -- Role of siRNAs in ESTABLISHMENT of transcriptional gene amplification -- silencing first discovered in plants de novo DNA methylation Simon and Meyers COPB 2011 PolIV and V in TGS tasiRNAs ,V Djupedal and Ekwall, Cell Research, 2009 PolIV and V in TGS PolIV PolV Wierzbicki et al. Cell, 2008 PolIV and V in TGS Wierzbicki et al. Cell, 2008 PolIV and V in TGS Wierzbicki et al. Cell, 2008 RNA methods of TGS (in plants) Haag and Pikaard, Nat. Rev. MCB, 2011 RNA methods of TGS (in plants) Maintenance spreading RNA methods of TGS (in plants) TGS speading A. thaliana RNA methods of TGS (in plants) In moss: role of miRNA in DNA methylation 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 Khraiwesh et al., Cell 2010 Role of siRNAs in silencing (plants) triggers DNA methylation recruitment of H3K9me role in maintenance of DNA methylation spreading RNA methods of TGS (S. pombe) Lejeune and Allshire, COCB 2011 RNA methods of TGS (S. pombe) 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 Potential for Inheritance? 1.siRNAs inherited, trigger H3K9me 2.Positive feedback between siRNAs and H3K9me: amplification and stabilization YES! PROTECTING THE GERMLINE piRNA Found in animals Role in germline Silencing of of repetitive DNA transposons, subtelomeric regions pericentromeric regions PROTECTING THE GERMLINE PIWI: role in piRNA generation Moazed Nature 2009 example of AGO activity Dicer independent PIWI-AGO 2ndary piRNA piRNA PROTECTING THE GERMLINE 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 Similar observations for female gametophyte Slotkin et al., Cell 2009 PROTECTING THE GERMLINE 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 Transcriptional Gene Silencing AGO-linked activities PolII PolIV TGS PTGS (post transcriptional gene silencing) message cleavage inhibition of translation (small and large ncRNAs) PTGS siRNA PTGS • RISC complex RNA-induced silencing complex • si RNA – AGO – DCR – dsRNA binding protein (TRBP) • miRNA – AGO – GW182 PTGS FEBS letters 2005 Systemic Silencing Small ncRNA movement some miRNAs can move (short distance) siRNAs can move: systemic responses 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?