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?