ppt presentation

- RNA interference
Discovery of RNA interference
- silencing of gene expression with dsRNA
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
What is the mechanism behind?
Original (!) hypotheses:
- antisense RNA mechanically prevents translation
- dsRNA is degraded (RNases)
Cosuppression in Petunia
Aim: increase expression of pigment-synthetizing enzym
Result: loss of pigmentation in flower segments
Napoli et al. 1990 Plant Cell 2:279–289
Cosuppression in Petunia
Expression of antisense RNA was less efficient!!!
Napoli et al. 1990 Plant Cell 2:279–289
Mechanism see later!
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. Hamilton, David C. Baulcombe*(1999):
A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants, Science 286 (5441): 950-952
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).
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
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
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)
Wang et al. 2004
• siRNA (small interfering) – from dsRNA of various origin (both
internal and external – thousands types (both in cis and in trans)
….. (+ piRNA in animals)
- 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)
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)
Mechanism of small RNA action - overview
Pol V
PTGS (21-22 nt): - specific cleavage of transcript
- block of translation
TGS (24 nt):
- methylation of promoter, heterochromatin formation
- preventing interaction of transcription factors
sRNA mode of action also depends
on complementarity
- incomplementarity in cleavage site prevents RNase activity
dsRNA formation
+ secondary structures of viral RNAs (!)
(MIR genes)
mRNA fragment after Ago
cleavage (secondarysiRNA )
• RdRP = RNA-dependent RNA Polymerase – synthesis of compl. RNA strand
- transcripts cleaved by RISC
- impaired mRNAs (without polyA or cap)
- transcripts of RNA polymerase IV
RNA polymerases in RNAi
(in plants)
RNA-dependent RNA polymerases (RdRP, RDR)
- form the majority siRNAs in plants
- 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
- dsRNA from products of RNA polymerase IV (DNA-dependent)
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
- 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
RNA-directed DNA methylation (in detail)
Pol IV a V – RNA polymerases
RDR2 - RNA dep.RNA polymerase
DCL3 – dicer-like protein
DRM2 – de novo metyltransferase
DRD1 – chromatin remodelling protein
SHH1 – dual histon-code reading
(H3K9me2, H3K4)
Saze et al. 2012
Zhang et al. 2013
- 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
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
Zhang et al. 2013
Zemach et al. 2013
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)
Cosupression in Petunia
- overexpression of pigment gene
(enzyme for pigment synthesis)
caused loss of pigmentation in flower
- 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
interaction (in trans) between homologous alleles (epialleles),
resulting in heritable change in gene expression of one allele
(= epimutation)
paramutagenic and
paramutated allele
mediator of
interaction (in trans) between homologous alleles (epialleles), resulting in heritable
change in gene expression of one allele (= epimutation)
1.Paramutagenic allele
2. Paramutable allele
- 1. methylated, inactive allele
transcribed by Pol IV
- siRNA formation
- 2. induction of RdDM of
complementary sequences
mediator of
MOP1 = ortholog RDR2
MOP2 = subunit of Pol IV, V
(mediator of paramutation)
RNAi pathways in plants - overview
+ Inverted repeat
Natural antisense
(DNA methylation)
Molnár et al. 2011
+ secondary siRNAs (theoretically from any RNA fragment primarily cleaved by Ago)
Antiviral systemic
- newly developed leaves resistant
against infection (1928)
siRNA are transported
- via plasmodesmata
- through phloem
- passive – diffusion or in direction of stream
Spreading of silencing
- GFP silencing with siRNAs (from vasculature)
green = GFP fluorescence, red = chlorophyll autofluorescence
Function of RNAi
and systemic spreading of sRNA
- antiviral defense – preventing of spreading of infection and new
- 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
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!)
Example: roles of miRNAs and ta-siRNAs in
Arabidopsis leaf development
Pulido A , Laufs P J. Exp. Bot. 2010;61:1277-1291
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 nonhomologous to miRNA)
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!
Parental imprinting
imprint establishment connected with meiosis
- extensive demethylation, specific methylation of some genes
imprint establishes during development of gametophyte
(haploid phase in plants: (polen) polen tube + embryo sac)
demethylation in a part of gametophyte – genetically terminal
- central cell of embryo sac (DME - DEMETER)
- vegetative nucleus of polen tube (block of DDM1, DME)
Epigenetic changes in microgametophyte
Inhibition of methylation (repression DDM1) + actively (DME)
In vegetative nucleus
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
Epigenetic changes in megagametophyte
Primary function:
- block of TE and endosperm specific genes
in egg cell
- regulation of endosperm size
- block of gametophytic developmental program?
Repression of gene expression independent
on methylation and siRNA:
- 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
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
VIN3 – deacethylation of histons, VRN2 – induction of H3K27 methylation
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
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