1. Epigenetics - RNA interference

2. Cenorhabditis elegans Discovery of RNA interference (1998) - silencing of gene expression with dsRNA

3. Antisense RNA (= RNA komplementární k mRNA) can silence gene expression (již počátek 80. let 20. století) - direct introduction of antisense RNA (or transcription in reverse orientation) - interaction of mRNA and antisense RNA, formation of dsRNA

4. What is the mechanism behind? Original (!) hypotheses: - antisense RNA mechanically prevents translation - dsRNA is degraded (RNases)

5. Cosuppression in Petunia Result: loss of pigmentation in flower segments Napoli et al. 1990 Plant Cell 2:279–289 Aim: increase expression of pigment-synthetizing enzym

6. Expression of antisense RNA was less efficient!!! Cosuppression in Petunia Napoli et al. 1990 Plant Cell 2:279–289 Mechanism see later!

7. What they get the Nobel prize for? - making proper controls pays off! - Introduction of even very small amount of dsRNA induce specific silencing ( antisence RNA is less efficient! ) dsRNA has to be a signal! - for sequence specific silencing Andrew J. HamiltonAndrew J. Hamilton, David C. Baulcombe*( 1999):David C. Baulcombe* A Species of Small Antisense RNA in Posttran- scriptional Gene Silencing in Plants, Science 286 (5441): 950-952

8. RNA interference (RNAi) = silencing of gene expression mediated by small RNAs (small RNA, sRNA) in plants predominantly - 21-24 nt The precise role of 25-nt RNA in PTGS remains to be determined. However, because they are long enough to convey sequence specificity yet small enough to move through plasmodesmata, it is possible that they are components of the systemic signal and specificity determinants of PTGS (Hamilton and Baulcombe, 1999).

9. RNA interference (RNAi) gene silencing at transcriptional level (TGS) (transcriptional gene silencing) - induction of DNA methylation (mRNA not formed) posttranscriptional level (PTGS) (posttranscriptional gene silencing) - transcript cleavage - block of translation

10. Basic mechanism of RNAi dsRNA in cell is cleaved by RNase DICER into short dsRNA fragments – sRNA Argonaute with a single strand (from sRNA) mediates recognision of complementary sequences, which should be silenced (TGS, PTGS)

11. Small RNA in plants - 3’ end of sRNA methylated (HEN1) - protection miRNA (micro) – from transcipts of RNA Pol II (pre-miRNA) – hunderds MIR genes (in trans) siRNA (small interfering) – from dsRNA of various origin (both internal and external – thousands types (both in cis and in trans) ….. (+ piRNA in animals) pre-miRNA Wang et al. 2004

12. Dicer-like - cleavage of dsRNA (21-24nt) - in Arabidopsis 4 paralogues (different functions) DCL1 – 21 nt miRNA (pre-miRNA) DCL3 – 24 nt siRNA (TGS - RdDM) DCL2, 4 – 21-22 nt siRNA (antiviral defence, secondary siRNAs)

13. Argonaute RNA binding protein (20-26 nt RNA) - strand selection (5’ nt, participation of HSP90) - 10 genes in Arabidopsis - main component of RISC (RNA induced silencing complex) - block of translation or slicer (RNAse H-like endonuclease - PIWI doména) - role in TGS (RdDM) (RNA directed DNA methylation)

14. Mechanism of small RNA action - overview PTGS (21-22 nt): - specific cleavage of transcript - - block of translation TGS (24 nt):- methylation of promoter, heterochromatin formation - preventing interaction of transcription factors Pol V

15. sRNA mode of action also depends on complementarity - incomplementarity in cleavage site prevents RNase activity

16. dsRNA formation (MIR genes) ? RdRP = RNA-dependent RNA Polymerase – synthesis of compl. RNA strand templates: - transcripts cleaved by RISC - impaired mRNAs (without polyA or cap) - transcripts of RNA polymerase IV + secondary structures of viral RNAs (!) mRNA fragment after Ago cleavage (secondarysiRNA )

17. RNA polymerases in RNAi (in plants) RNA-dependent RNA polymerases (RdRP, RDR) - form the majority siRNAs in plants RDR6 : - dsRNA from impaired (not by Ago) RNAs (lacking polyA or cap) = primary siRNAs - dsRNA from products of Ago = secondary siRNAs - RDR1 – close paralogue involved in antiviral defense RDR2 : - dsRNA from products of RNA polymerase IV (DNA-dependent)

18. RNA polymerases in RNAi (in plants) DNA dependent RNA polymerase (Pol IV and Pol V) - related to RNA polymerase II (share common subunits) - specific for plants RNA polymerase IV - transcibes chromatin with H3K9me2 and non-met H3K4 (SHH1) - short transcripts (hundreds nt), with cap, without polyA - automatically used by RDR2 (  dsRNA  DCL3  siRNA) RNA polymerase V - necessary for RNA-directed DNA methylation (RdDM) - direct interaction with Ago4 – identification of target seq. RNA polymeráza II – unclear, but important role

19. RNA-directed DNA methylation (in detail) - methylation (by DRM2) occurs only under interaction of AGO4(6,9)-siRNA with RNA Pol V ( large subunit C-term domain ) and RNA-RNA complementarity Pol IV a V – RNA polymerases RDR2 - RNA dep.RNA polymerase DCL3 – dicer-like protein AGO4 – ARGONAUTE DRM2 – de novo metyltransferase DRD1 – chromatin remodelling protein SHH1 – dual histon-code reading (H3K9me2, H3K4) Saze et al. 2012 SHH1 Zhang et al. 2013

20. RNA-directed DNA methylation – why so complicated and energy consuming? - de novo methylation of TE in new insertion sites - transmission of info from histons (CMT2/3) less reliable (less dense nucleosomes) - PTGS – TGS transition SHH1 Zhang et al. 2013 Zemach et al. 2013

21. Secondary siRNA formation - target RNA (mRNA, TAS transcripts) - cleaved by Ago + primary sRNA (miRNA or siRNA) - RDR6 – complementary strand synthesis: dsRNA  DCL2(4)  secondary siRNA Function of secondary siRNA - signal amplification - formation of siRNA from neighbor seq. (transitivity – new targets) ta-siRNAs (miRNA na TAS) ( trans-acting siRNA – widening of miRNA targets)

22. - overexpression of pigment gene (enzyme for pigment synthesis) caused loss of pigmentation in flower sectors Cosupression in Petunia - occurrence of aberrant transcripts due to overexpression - formation of dsRNA from aberrant transcripts by RdRP (RDR6) - formation of siRNAs that silence both transgene and internal gene Use in functional genomics – overexpression and knock-out possible with a single gene construct dsRNA

23. Paramutation interaction (in trans) between homologous alleles (epialleles), resulting in heritable change in gene expression of one allele (= epimutation) paramutagenic and paramutated allele Mechanism? mediator of paramutation

24. Paramutation interaction (in trans) between homologous alleles (epialleles), resulting in heritable change in gene expression of one allele (= epimutation) - 1. methylated, inactive allele transcribed by Pol IV - siRNA formation - 2. induction of RdDM of complementary sequences 1.Paramutagenic allele 2. Paramutable allele MOP1 = ortholog RDR2 MOP2 = subunit of Pol IV, V (mediator of paramutation) mediator of paramutation

25. RNAi pathways in plants - overview Natural antisense + Inverted repeat Molnár et al. 2011 (DNA methylation) a b c d + secondary siRNAs (theoretically from any RNA fragment primarily cleaved by Ago)

26. Antiviral systemic resistance - newly developed leaves resistant against infection (1928) Mechanism?

27. siRNA are transported - via plasmodesmata - through phloem - passive – diffusion or in direction of stream

28. Spreading of silencing - GFP silencing with siRNAs (from vasculature) green = GFP fluorescence, red = chlorophyll autofluorescence

29. Function of RNAi and systemic spreading of sRNA - antiviral defense – preventing of spreading of infection and new infection - TE defense – keeping genome integrity, heterochromatin structure - regulation of development – practically all phases - stress response (even heritable changes – see bellow) - regulation of nutrient uptake – e.g. phosphorus - epigenetic modulation of genetic information in meristems (heritable changes) - environmental adaptations

30. Epigenetic regulation of gene expression in plant development - regulation mainly with miRNA (21 nt) (hundreds of MIR genes) - sometimes mediated with ta-siRNA (non-coding TAS transcript cleaved by RISC with miRNA, RDR6, …) - frequently transported between neighbour cells (non-cell autonomous) - targets – genes for regulatory proteins e.g. transcription factors, components of ubiquitination pathway, … (limited reliability of simple promoter fusion experiments with such genes!)

31. Example: roles of miRNAs and ta-siRNAs in Arabidopsis leaf development Pulido A, Laufs P J. Exp. Bot. 2010;61:1277-1291

32. Modulation of RNAi regulation of gene expression in plant development Phenotypic changes caused by knock-out of a MIR gene Phenotypic changes caused by expression of miRNA-resistant variants of target genes (same protein encoded by different nucleotide sequence non- homologous to miRNA)

33. Parental imprinting - varying epigenetic modification of alleles inherited from father and mather (parental imprint) – it results in differencial expression of these alleles in zygote = alleles of some genes are methylated in one or the other gamet - evolved in mammals and angiospermous plants - hemizygote“ state can serve to ensure reasonable feeding of embryo (in mather body) – prevents evolution of paternal alleles causing excessive embryo feeding (in angiosperms – determination of endosperm size) - in plants – main function – repression of TE activity!

34. Parental imprinting Mammals: imprint establishment connected with meiosis - extensive demethylation, specific methylation of some genes Plants: imprint establishes during development of gametophyte (haploid phase in plants: (polen) polen tube + embryo sac) demethylation in a part of gametophyte – genetically terminal lines- central cell of embryo sac (DME - DEMETER) - vegetative nucleus of polen tube (block of DDM1, DME)

35. Epigenetic changes in microgametophyte Primary function: induction of proper TE methylation in generative cells (sperm cells): Demethylation – reactivation of TE – siRNA formation – transport to spermatic cells – induction of proper TE methylation Inhibition of methylation (repression DDM1) + actively (DME) In vegetative nucleus

36. Primary function: - block of TE and endosperm specific genes in egg cell - regulation of endosperm size - block of gametophytic developmental program? Epigenetic changes in megagametophyte

37. - repression state kept primarily by di-(tri-)methylation of histon H3K27 - in vegetative Arabidopsis tissue – thousands genes! - Maintenance of H3K27me2 mediated by gene specific Polycomb repressive complex (PRC2) - keep histon methylation after replication Repression of gene expression independent on methylation and siRNA: PcG (POLYCOMB GROUP) proteins

38. Example: vernalization VIN3 – deacethylation of histons, VRN2 – induction of H3K27 methylation after vernalization VRN2 (VERNALIZATION) PcG protein binds with other proteins to promoter of FLC gene (repression by H3K27me2), expression of FLC blocks flowering - Repression of FLC allows formation of generative organs

39. Vernalization – primary signal? VIN3 chromatin: - permanently H3K27me3 repressive mark (PRC2) - transcription induces activation mark H3K36me3 and acethylation (? Hypothetically low temperature induce disassembly of nucleosome in TSS?) - bivalent chromatin labeling (  animal stem cells) - quantitative response