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An introduction to RNAi
technology
OECD Conference on Regulation of Externally Applied
dsRNA-based Products for Management of Pests
10.-12.4. 2019, Paris
Petr Svoboda
Institute of Molecular Genetics AS CR, Prague
response(s) to
double-stranded RNA
Sources & types of dsRNA
Exogenous
viral life cycle
inverted repeats convergent transcription pairing in trans
Endogenous
• frequently associated with parasitic mobile elements
genome
dsRNA response
adenosine
deamination
interferon
response
innate immunity
SEQUENCE-INDEPENDENT
RNAi
innate immunity
genome defense
SEQUENCE-SPECIFIC
dsRNA
dsRBD
dsRNA binding domain
other
e.g. Staufen-mediated decay
RNA silencing
RNAi & related pathways
• the introduced gene created a block in
anthocyanin biosynthesis.
• variable color phenotype co-segregated
with the introduced CHS gene
• mRNA of endogenous CHS was reduced
50-fold from wild-type levels
• the erratic and reversible nature of this
phenomenon suggests the possible
involvement of methylation.
The Beginning
uninjected parentnot stained
antisense injected
mex-3insituhybridization
dsRNA injected
Canonical animal RNAi pathway
The principle of RNA silencing
substrate
Dicersmall RNA
production
silencing
(function)
RdRP
AGO
targeting
AGO
RdRP
amplificiation
RdRP
Dicer
AGO
AGO
AAAAA
mRNA cleavage
siRNA
dsRNA
siRNA
dsRBP
aberrant RNA
AGO
dicing
slicing
RISC
loading
substrate
synthesis
viruses, convergent transcription, inverted
repeat transcription, RdRp activity, artificial
Canonical animal RNAi pathway
• defense against elements (viruses, TEs) producing dsRNA
• with or without RdRP amplification loop
RNAi: long dsRNA-induced sequence-specific mRNA degradation
dicing
repression
RISC
(miRISC)
loading miRNA
Dicer
AGO
AAAAA
inhibition of translation
mRNA degradation
GW182
pre-miRNA
dsRBP
AGO
Drosha
pri-miRNApre-miRNA
DGCR8
Microprocessor complex
EXP5
mRNA degradation
RELOCATION TO
P-BODIES
nucleus
cytoplasm
Canonical animal miRNA pathway
DGCR8
• post-transcriptional regulation of gene expression
• stoichiometry (miRNA abundance/cell) matters
miRNA: regulatory RNAs produced by Dicer from genome-encoded
small hairpin precursors
DICER
small RNA biogenesis
Dicer
RNaseIIIa“platform”
(DUF283)
RNaseIIIb
PAZ
DEXD
HELICc
NSMB 2012 19(4):436-40.
Dicer
• Dicer makes small RNAs
Three distinct small RNA populationsDicer
• predictability of the 5’ end
ARGONAUTE
small RNA loading
target recognition
silencing
Argonaute-mediated silencing effectsAGO
AGO2
AAAAA
mRNA cleavage by AGO2
Eukaryots
“miRNA-like” mRNA
degradation
P-BODIES
CCR4-NOT
AGO
AAAAA
Inhibition of translation/deadenylation
GW182
Eukaryots
“RNAi-like”
RdDM
DNA
methylation
Plants
other transcriptional silencing
heterochromatin
e.g. S. pombe … poorly understood in animals
POST-TRANSCRIPTIONAL
TRANSCRIPTIONAL
Argonaute structure and functionAGO
PIWI
PAZ
N
MID
mRNA
siRNA
3’
5’
5’3’
• Argonaute structure explains principles of target recognition
and repression
Argonaute structure and functionAGO
PIWI
PAZ
N
MID
mRNA
siRNA
3’
5’
5’3’
• Dissociation constants for seed matching targets are in a pM range
• low abundant miRNAs unlikely to have significant regulatory effects
• seed match + abundance = siRNA off-targeting
Argonaute-mediated silencing effectsAGO
Parameters influencing silencing by small RNAs
• small RNA abundance (stoichiometry)
• target site accessibility
• complementarity with the target
• type of silencing (transcriptional/post-transcriptional)
Small RNA seed sequence defines the minimal
sequence complementarity required for silencing.
OFF-TARGETING
Perfect vs. imperfect
basepairing
nucleus cytoplasm
Ago
Class 2 hairpin
(miRNA-like)
Class1 hairpin
(shRNA)
siRNA
EXPRESSION VECTOR
.
TRANSFECTION
Ago2
AAAAA
Cleavage of
mRNA by Ago2
Ago
AAAAA
mRNA degradation Inhibition of translation
RELOCATION TO
P-BODIES
RISC
loading
short RNAs
(miRNAs and siRNAs)
Argonaute – targeting & off-targetingAGO
• off-targeting is siRNA-specific
• any siRNA has off-targeting potential
Jackson et al. (2003)
Nature Biotech
Argonaute – targeting & off-targetingAGO
Argonaute – targeting & off-targetingAGO
Jackson et al. (2003)
Nature Biotech
• off-targeting is largely concentration-dependent
• it is strongly reduced in sub-nanomolar range
transfected at 100nM
siRNA2 siRNA4 siRNA3 siRNA1 pool
Thermofisher/Dharmacom website
Argonaute – targeting & off-targetingAGO
• siRNA pooling is a way to reduce concentrations of individual
siRNAs while keeping the constant siRNA amount in a transfection
• natural siRNA pools produced from siRNAs are highly specific
because of a highly diluted off-targeting effect
Argonaute – targeting & off-targetingAGO
• off-targeting potential stems from seed sequence frequency
• siRNA knock-downs - usually employ nM concentrations
• hydrodynamic transfection (40 mg/mouse – Nature, 418, 38-39)
Ago
AAAAA
miRNA-like
Inhibition
of translation
seed = nucleotides 2-7
Ago2
AAAAA
RNAi-like
Cleavage of
mRNA by Ago2
seed = nucleotides 2-7
RNA interference
in different
multicellular
organisms
mRNA
degradation
RNAi
Dicer
Co-existence of miRNA & RNAi pathways
defensegene control
inhibition
of translation
miRNA
Dicer
AGO AGO
Arthropod set up
mRNA
degradation
RNAi
Co-existence of miRNA & RNAi pathways
defensegene control
inhibition
of translation
miRNA
AGO
Dicer
Annelida set up
(some Molluscs?)
mRNA
degradation
RNAi
defensegene control
inhibition
of translation
miRNA
AGO
Dicer
interferon
response
PKR
Co-existence of miRNA & RNAi pathways
defense
Vertebrate set up
mRNA
degradation
RNAi
Co-existence of miRNA & RNAi pathways
defensegene control
inhibition
of translation
miRNA
AGO
Dicer
RdRP
Nematode set up
(some Molluscs?)
Nematodes
endoRNAi antiviral
defense
replication
dsRNA dsRNA
DRH-1
Dicer
gene control
ERGO-1
AAAAA
mRNA cleavage
RdRP
WAGOs
RDE-4
1o siRNA
1o siRNA
26G RNA
22G RNA
DRH-3
2o siRNA
Dicer
immunity
RDE-1
AAAAA
mRNA cleavage
RdRP
SAGO-2
RDE-4
1o siRNA
22G RNA
DRH-3
2o siRNA
ERI
ERI
RDE-8
1o siRNA
22-23 nt
RNA clearance
exoRNAi
RDE-1
AAAAA
mRNA cleavage
RDE-8
RdRP
WAGOs
dsRNA
injection
feeding
soaking
1o siRNA
1o siRNA
22-23 nt
22G RNA
DRH-3
2o siRNA
DRH-1
Dicer
RDE-4
nucleus
cytoplasm
Dicer
ALG-1/2
gene control
AGO
AAAAA
inhibition of translation
AIN-1
pre-miRNA
miRNA
miRNA
22-23 nt
pri-miRNA
Microprocessor
complex
Plants
RDR6
RNA clearance
(post-transcriptional)
transgene & viral silencing
AGO
dsRNA
viral
long hairpin
21/22 nt siRNA
SDE3
sense RNA
RDR6
SGS3
DCL4/2 DCL3
AGO4/6
24 nt siRNA
RdDM
AGO
SDE3
DNA methylation
(transcriptional)
HEN1 HEN1DRB3DRB4
sense RNA
TAS loci
AGO
miRNA
RDR6
DCL4
AGO1/7
tasiRNA
21nt tasiRNA
Gene regulation during
development
HEN1DRB
Plants
AGO1
DCL2 DCL3
AGO10AGO7
DCL1
miR-390 miR-156/166
U UAA
AGO2
MAIN miRNA PATHWAY
AGO4/6/9
21 nt 24 nt
long inverted repeats
(evolving miRNAs)
ALTERNATIVE miRNA PATHWAY
DCL4
• highly complex RNA silencing system 4x Dicer, 10-20 Argonautes
• a number of small RNAs, TGS & PTGS effects
Plants – transcriptional silencing
Canonical RdDM Non-canonical RdDM
• highly complex RNA silencing – crosstalks & redundancy
Key points
• a targeting repertoire of a small RNA is largely determined by its
seed – nucleotides 2-8.
• not absolute rule (non-canonical binding)
• allows some predictability, especially for conserved targets
• RNAi-like cleavage or miRNA-like target repression silencing
effects are primarily defined by AGO isoform and basepairing
• targeting efficiency is determined by:
• small RNA abundance (stoichiometry)
• target site accessibility
• complementarity with the target
• vertebrates have lack systemic RNAi, an RdRP amplification
system, and highly processive Dicer -> inefficient RNAi
• plant small RNA pathways use 3’ end 2-O-methyl modification of
all small RNAs. In mammals, such modification is found only in
piRNAs bound to PIWI AGO clade in the germline
STAY TUNED
AGO
Dicer
Dicer AGO
dicing
RISC-loading complex
asymmetry sensing
HSP90
AGO
Dicer
HSP90
Argonaute loading passenger strand
removal
Argonaute loadingAGO
sense siRNA strand (passenger)
antisense siRNA
= targeting (active) strand!
5’-CGUACGCGGAAUACUUCGAdTdT-3’
|||||||||||||||||||
3’-dTdTGCAUGCGCCUUAUGAAGCU-5’
• siRNA duplex undergoes loading of one of the strands on RISC
• 5' portion of the selected strand is paired less stably than its 3' portion
• ssRNA could reconstitute RISC; 10- to 100-fold higher concentrations required
relative to siRNA duplexes (Martinez et al., 2002, Cell. 110(5):563-74)
RNAi mobility - systemic RNAi
dsRNA
dsRNA
dsRNA
dsRNA delivery RNAi effect
Cell autonomous
RNAi
Systemic
RNAi
Environmental
RNAi
dsRNA
Example
0.5 - 1.0x106 dsRNA molecules per each gonad arm
mammals
C. elegans
some Arthropods (Tribolium)
plants
C. elegans
insects
Nematodes
C. elegans is an outstanding model for analyzing RNA silencing
• highly complex RNA silencing system
• one Dicer but 26 Argonautes and 3 RdRPs
• four pathways can be recognized
• miRNA
• exoRNAi
• endoRNAi
• antiviral defense
• primary and secondary RNAs (amplification of the response)
• cytoplasmic and nuclear Argonautes
• systemic RNAi, sensitive, cheap, temperate areas worldwide
0.5 - 1.0x106 dsRNA molecules per each gonad armTabara et al., 1998
Nematodes
RNA clearance
exoRNAi
RDE-1
AAAAA
mRNA cleavage
RDE-8
RdRP
WAGOs
dsRNA
injection
feeding
soaking
1o siRNA
1o siRNA
22-23 nt
22G RNA
DRH-3
2o siRNA
DRH-1
Dicer
RDE-4
0.5 - 1.0x106 dsRNA molecules per each gonad arm
Tabara et al., 1998
miRNA pathway in plants & animals
gene control
Plants
mRNA cleavage
inhibition of translation
pre-miRNA
HYL1
AGO1miRNA
21 nt
pri-miRNA
SE
HYL1SE
DCL1
DCL1
AGO1
AAAAA
SUO
HEN1HEN1
nucleus
cytoplasm
nucleus
cytoplasm
Arthropods
AGO1
gene control
AGO1
AAAAA
inhibition of translation
GW182
LOQS
miRNA
21-23 nt
Dicer-1
nucleus
cytoplasm
Mammals
AGO1-4
gene control
AGO1-4
AAAAA
inhibition of translation
GW182
TARBP2
miRNA
21-23 nt
Dicer-1
pre-miRNA
pri-miRNA
pre-miRNA
pri-miRNA
DGCR8
DGCR8
Drosha DGCR8
DGCR8
Drosha
Plants – spreading of RNA silencing
short distance
long distance
Plants –> Animals


?
?
?
environmental
& systemic RNAi
circulating
miRNAs?
?
Huang et al., 2006
Baum et al., 2007
Mao et al. 2007
environmental
& systemic RNAi
Plants –> Animals
Unclear/controversial issues:
Mechanism of transport
• Mechanism of transport across membranes not explained
• Unclear if free or bound to a protein
• Survival in digestive tract?
Effector complex structure
• Would require binding of methylated single stranded RNAs by AGO
Targeting stoichiometry
• Concentrations estimated 68-250 fM – too low
• Authors calculate ~850 molecules per cell, cannot be verified – data not released
Plants –> Animals
• meta-study of xenomiRs of 824 datasets from human tissues and body fluids
• xenomiRs commonly present in tissues (17%) and body fluids (69%),
• low abundance, 0.001% of host human miRNA counts
• no significant enrichment in sequencing data from tissues and body fluids exposed
to dietary intake (e.g. liver).
• no significant depletion in tissues and body fluids that are relatively separated
from the main bloodstream (e.g, brain and cerebro-spinal fluids)
• the majority (81%) of body fluid xenomiRs stem from rodents, which are rare
human dietary contributions, but common laboratory animals.
• body fluid samples from the same studies are clustered by xenomiR compositions
- suggesting technical batch effects.
• feeding studies - no transfer of plant miRNAs into rat blood, or bovine milk
sequences into piglet blood.
doi: 10.1261/rna.059725.116
RNA, advanced online, Jan 6., 2017
Animal Dicer evolution
• RNAi-dedicated Dicer-2 in Arthropods is a
derived character
• the mammalian “miRNA” Dicer is related
to miRNA-producing Dicer-1 in Arthropods
• Dicer in C. elegans produces efficiently
miRNAs and siRNAs
Dicer
“miRNA” Dicer
nucleus cytoplasm
GW182
AGO2
AAAAA
Cleavage of
mRNA by Ago2
Exportin 5-mediated
transport
AGO
AAAAA
mRNA degradation
Inhibition of translation
relocation
to P-bodies
GW182
miRNA
duplex
Dicer
GW182
AGO
RISC
loading
Mammalian microRNA pathway
pri-miRNA pre-miRNA
DGCR8
Microprocessor
complex
DGCR8
Dicer cleavage
targeting
pre-miRNA
Drosha
miRISC
Mammals (and vertebrates in general)
OAS
MDA5
TLR3
PACT
Dicer
miRNA RNAi
AGO1-4
gene control
AGO1-4
AAAAA
inhibition of translation
GW182
AGO2
AAAAA
mRNA cleavage
pre-miRNA
miRNA
dsRNA
siRNA
TARBP2
PKR
OAS
RIG-I
translational
repression
RNAse L
IFN signaling
interferons
&
interferon stimulated
genes
Interferon
response
common sensors
RNA silencing
antiviral defense
dsRNA
• miRNA pathway is the main RNA silencing pathway
• main dsRNA response = sequence-independent interferon response
Annelids
Dicer
miRNA RNAi
AGO
AGO
AAAAA
inhibition of translation
AGO
AAAAA
mRNA cleavage
pre-miRNA
miRNA
dsRNA
siRNA
TARBP2
?
OAS
RIG-I ?
MDA5 ?
RNAse L
signaling
innate immunity?
dsRNA response
common sensors
RNA silencing
dsRNA
?
?
• almost no functional data, set-up seems similar to mammals
Molluscs
RdRP
Dicer
miRNA RNAi
AGO
gene control & antiviral defense?
AGO
AAAAA
inhibition of translation
GW182
AGO
AAAAA
mRNA cleavage
pre-miRNA
miRNA
dsRNA
siRNA
TARBP2
PKR
OAS
RIG-I
MDA5
translational
repression
RNAse L
IFN signaling
interferons
&
interferon stimulated
genes
Interferon
response
common sensors
RNA silencing
antiviral defense
dsRNA
? MX
• almost no functional data, set-up seems similar to mammals
• possible RdRP loop – would make it similar to nematodes
Arthropods
nucleus
cytoplasm
AGO2
defense
gene control
AGO2
AAAAA
mRNA cleavage
siRNA
dsRNA
PKR
RIG-I
MDA5
Interferon
response
common sensors
RNAi
dsRNA
AGO1
gene control
AGO1
AAAAA
inhibition of translation
GW182
pre-miRNA
LOQS
miRNA
miRNA
21-23 nt
pri-miRNA
Microprocessor
R2D2
Dicer-1
Dicer-2
TLR3?
signaling
innate immunity?
• separated miRNA & RNAi
• sensors of the interferon response present
Interferon response induced by long dsRNA (>30bp)
sensing
specific
responses
MDA5 TLR3
PKR
OAS
RIG-I
common
response
INTERFERON RESPONSE
ISG
interferon-stimulated genes
eIF2a
P
RNaseL
global inhibition
of translation
global mRNA
degradation
2’,5’-OA
• The interferon response can be detected/monitored
lack of 3’ overhangs
induces IFN via Rig-I
dsRNA > 30 bp activates PKR and 2’,5’-OAS
some sequence
motifs within ssRNA
can activate IFN
cationic lipid-RNA complexes
activate IFN via TLR3 and TLR7
5’ triphosphate introduced by
phage RNA polymerases
activates IFN
siRNA < 30 bp can activate PKR
lack of 3’ overhangs
induces IFN via Rig-I
dsRNA > 30 bp activates PKR and 2’,5’-OAS
some sequence
motifs within ssRNA
can activate IFN
cationic lipid-RNA complexes
activate IFN via TLR3 and TLR7
5’ triphosphate introduced by
phage RNA polymerases
activates IFN
siRNA < 30 bp can activate PKR
Interferon response induced by siRNAs
24hours72hours
mock
siRNA A
siRNA B1
siRNA B2
siRNA C
mock
siRNA A
siRNA B1
siRNA B2
siRNA C
dsRBDZβ deaminaseZα
NLSNES
ADAR1p150
ADAR1p110
ADAR2
ADAR3
Adenosine deamination
Kono & Akiyama, 2013
DOI: 10.5772/55203
nucleus
cytoplasm
ADAR1
degradation
?
Rana 2007

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An introduction to RNAi technology - Petr Svoboda - Institute of Molecular Genetics of the ASCR, Czech Republic

  • 1. An introduction to RNAi technology OECD Conference on Regulation of Externally Applied dsRNA-based Products for Management of Pests 10.-12.4. 2019, Paris Petr Svoboda Institute of Molecular Genetics AS CR, Prague
  • 3. Sources & types of dsRNA Exogenous viral life cycle inverted repeats convergent transcription pairing in trans Endogenous • frequently associated with parasitic mobile elements genome
  • 4. dsRNA response adenosine deamination interferon response innate immunity SEQUENCE-INDEPENDENT RNAi innate immunity genome defense SEQUENCE-SPECIFIC dsRNA dsRBD dsRNA binding domain other e.g. Staufen-mediated decay
  • 5. RNA silencing RNAi & related pathways
  • 6. • the introduced gene created a block in anthocyanin biosynthesis. • variable color phenotype co-segregated with the introduced CHS gene • mRNA of endogenous CHS was reduced 50-fold from wild-type levels • the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation. The Beginning
  • 7. uninjected parentnot stained antisense injected mex-3insituhybridization dsRNA injected Canonical animal RNAi pathway
  • 8. The principle of RNA silencing substrate Dicersmall RNA production silencing (function) RdRP AGO targeting AGO RdRP amplificiation
  • 9. RdRP Dicer AGO AGO AAAAA mRNA cleavage siRNA dsRNA siRNA dsRBP aberrant RNA AGO dicing slicing RISC loading substrate synthesis viruses, convergent transcription, inverted repeat transcription, RdRp activity, artificial Canonical animal RNAi pathway • defense against elements (viruses, TEs) producing dsRNA • with or without RdRP amplification loop RNAi: long dsRNA-induced sequence-specific mRNA degradation
  • 10. dicing repression RISC (miRISC) loading miRNA Dicer AGO AAAAA inhibition of translation mRNA degradation GW182 pre-miRNA dsRBP AGO Drosha pri-miRNApre-miRNA DGCR8 Microprocessor complex EXP5 mRNA degradation RELOCATION TO P-BODIES nucleus cytoplasm Canonical animal miRNA pathway DGCR8 • post-transcriptional regulation of gene expression • stoichiometry (miRNA abundance/cell) matters miRNA: regulatory RNAs produced by Dicer from genome-encoded small hairpin precursors
  • 13. Three distinct small RNA populationsDicer • predictability of the 5’ end
  • 14. ARGONAUTE small RNA loading target recognition silencing
  • 15. Argonaute-mediated silencing effectsAGO AGO2 AAAAA mRNA cleavage by AGO2 Eukaryots “miRNA-like” mRNA degradation P-BODIES CCR4-NOT AGO AAAAA Inhibition of translation/deadenylation GW182 Eukaryots “RNAi-like” RdDM DNA methylation Plants other transcriptional silencing heterochromatin e.g. S. pombe … poorly understood in animals POST-TRANSCRIPTIONAL TRANSCRIPTIONAL
  • 16. Argonaute structure and functionAGO PIWI PAZ N MID mRNA siRNA 3’ 5’ 5’3’ • Argonaute structure explains principles of target recognition and repression
  • 17. Argonaute structure and functionAGO PIWI PAZ N MID mRNA siRNA 3’ 5’ 5’3’ • Dissociation constants for seed matching targets are in a pM range • low abundant miRNAs unlikely to have significant regulatory effects • seed match + abundance = siRNA off-targeting
  • 18. Argonaute-mediated silencing effectsAGO Parameters influencing silencing by small RNAs • small RNA abundance (stoichiometry) • target site accessibility • complementarity with the target • type of silencing (transcriptional/post-transcriptional) Small RNA seed sequence defines the minimal sequence complementarity required for silencing.
  • 20. nucleus cytoplasm Ago Class 2 hairpin (miRNA-like) Class1 hairpin (shRNA) siRNA EXPRESSION VECTOR . TRANSFECTION Ago2 AAAAA Cleavage of mRNA by Ago2 Ago AAAAA mRNA degradation Inhibition of translation RELOCATION TO P-BODIES RISC loading short RNAs (miRNAs and siRNAs) Argonaute – targeting & off-targetingAGO
  • 21. • off-targeting is siRNA-specific • any siRNA has off-targeting potential Jackson et al. (2003) Nature Biotech Argonaute – targeting & off-targetingAGO
  • 22. Argonaute – targeting & off-targetingAGO Jackson et al. (2003) Nature Biotech • off-targeting is largely concentration-dependent • it is strongly reduced in sub-nanomolar range
  • 23. transfected at 100nM siRNA2 siRNA4 siRNA3 siRNA1 pool Thermofisher/Dharmacom website Argonaute – targeting & off-targetingAGO • siRNA pooling is a way to reduce concentrations of individual siRNAs while keeping the constant siRNA amount in a transfection • natural siRNA pools produced from siRNAs are highly specific because of a highly diluted off-targeting effect
  • 24. Argonaute – targeting & off-targetingAGO • off-targeting potential stems from seed sequence frequency • siRNA knock-downs - usually employ nM concentrations • hydrodynamic transfection (40 mg/mouse – Nature, 418, 38-39) Ago AAAAA miRNA-like Inhibition of translation seed = nucleotides 2-7 Ago2 AAAAA RNAi-like Cleavage of mRNA by Ago2 seed = nucleotides 2-7
  • 26. mRNA degradation RNAi Dicer Co-existence of miRNA & RNAi pathways defensegene control inhibition of translation miRNA Dicer AGO AGO Arthropod set up
  • 27. mRNA degradation RNAi Co-existence of miRNA & RNAi pathways defensegene control inhibition of translation miRNA AGO Dicer Annelida set up (some Molluscs?)
  • 29. mRNA degradation RNAi Co-existence of miRNA & RNAi pathways defensegene control inhibition of translation miRNA AGO Dicer RdRP Nematode set up (some Molluscs?)
  • 30. Nematodes endoRNAi antiviral defense replication dsRNA dsRNA DRH-1 Dicer gene control ERGO-1 AAAAA mRNA cleavage RdRP WAGOs RDE-4 1o siRNA 1o siRNA 26G RNA 22G RNA DRH-3 2o siRNA Dicer immunity RDE-1 AAAAA mRNA cleavage RdRP SAGO-2 RDE-4 1o siRNA 22G RNA DRH-3 2o siRNA ERI ERI RDE-8 1o siRNA 22-23 nt RNA clearance exoRNAi RDE-1 AAAAA mRNA cleavage RDE-8 RdRP WAGOs dsRNA injection feeding soaking 1o siRNA 1o siRNA 22-23 nt 22G RNA DRH-3 2o siRNA DRH-1 Dicer RDE-4 nucleus cytoplasm Dicer ALG-1/2 gene control AGO AAAAA inhibition of translation AIN-1 pre-miRNA miRNA miRNA 22-23 nt pri-miRNA Microprocessor complex
  • 31. Plants RDR6 RNA clearance (post-transcriptional) transgene & viral silencing AGO dsRNA viral long hairpin 21/22 nt siRNA SDE3 sense RNA RDR6 SGS3 DCL4/2 DCL3 AGO4/6 24 nt siRNA RdDM AGO SDE3 DNA methylation (transcriptional) HEN1 HEN1DRB3DRB4 sense RNA TAS loci AGO miRNA RDR6 DCL4 AGO1/7 tasiRNA 21nt tasiRNA Gene regulation during development HEN1DRB
  • 32. Plants AGO1 DCL2 DCL3 AGO10AGO7 DCL1 miR-390 miR-156/166 U UAA AGO2 MAIN miRNA PATHWAY AGO4/6/9 21 nt 24 nt long inverted repeats (evolving miRNAs) ALTERNATIVE miRNA PATHWAY DCL4 • highly complex RNA silencing system 4x Dicer, 10-20 Argonautes • a number of small RNAs, TGS & PTGS effects
  • 33. Plants – transcriptional silencing Canonical RdDM Non-canonical RdDM • highly complex RNA silencing – crosstalks & redundancy
  • 34. Key points • a targeting repertoire of a small RNA is largely determined by its seed – nucleotides 2-8. • not absolute rule (non-canonical binding) • allows some predictability, especially for conserved targets • RNAi-like cleavage or miRNA-like target repression silencing effects are primarily defined by AGO isoform and basepairing • targeting efficiency is determined by: • small RNA abundance (stoichiometry) • target site accessibility • complementarity with the target • vertebrates have lack systemic RNAi, an RdRP amplification system, and highly processive Dicer -> inefficient RNAi • plant small RNA pathways use 3’ end 2-O-methyl modification of all small RNAs. In mammals, such modification is found only in piRNAs bound to PIWI AGO clade in the germline
  • 36. AGO Dicer Dicer AGO dicing RISC-loading complex asymmetry sensing HSP90 AGO Dicer HSP90 Argonaute loading passenger strand removal Argonaute loadingAGO sense siRNA strand (passenger) antisense siRNA = targeting (active) strand! 5’-CGUACGCGGAAUACUUCGAdTdT-3’ ||||||||||||||||||| 3’-dTdTGCAUGCGCCUUAUGAAGCU-5’ • siRNA duplex undergoes loading of one of the strands on RISC • 5' portion of the selected strand is paired less stably than its 3' portion • ssRNA could reconstitute RISC; 10- to 100-fold higher concentrations required relative to siRNA duplexes (Martinez et al., 2002, Cell. 110(5):563-74)
  • 37. RNAi mobility - systemic RNAi dsRNA dsRNA dsRNA dsRNA delivery RNAi effect Cell autonomous RNAi Systemic RNAi Environmental RNAi dsRNA Example 0.5 - 1.0x106 dsRNA molecules per each gonad arm mammals C. elegans some Arthropods (Tribolium) plants C. elegans insects
  • 38. Nematodes C. elegans is an outstanding model for analyzing RNA silencing • highly complex RNA silencing system • one Dicer but 26 Argonautes and 3 RdRPs • four pathways can be recognized • miRNA • exoRNAi • endoRNAi • antiviral defense • primary and secondary RNAs (amplification of the response) • cytoplasmic and nuclear Argonautes • systemic RNAi, sensitive, cheap, temperate areas worldwide 0.5 - 1.0x106 dsRNA molecules per each gonad armTabara et al., 1998
  • 39. Nematodes RNA clearance exoRNAi RDE-1 AAAAA mRNA cleavage RDE-8 RdRP WAGOs dsRNA injection feeding soaking 1o siRNA 1o siRNA 22-23 nt 22G RNA DRH-3 2o siRNA DRH-1 Dicer RDE-4 0.5 - 1.0x106 dsRNA molecules per each gonad arm Tabara et al., 1998
  • 40. miRNA pathway in plants & animals gene control Plants mRNA cleavage inhibition of translation pre-miRNA HYL1 AGO1miRNA 21 nt pri-miRNA SE HYL1SE DCL1 DCL1 AGO1 AAAAA SUO HEN1HEN1 nucleus cytoplasm nucleus cytoplasm Arthropods AGO1 gene control AGO1 AAAAA inhibition of translation GW182 LOQS miRNA 21-23 nt Dicer-1 nucleus cytoplasm Mammals AGO1-4 gene control AGO1-4 AAAAA inhibition of translation GW182 TARBP2 miRNA 21-23 nt Dicer-1 pre-miRNA pri-miRNA pre-miRNA pri-miRNA DGCR8 DGCR8 Drosha DGCR8 DGCR8 Drosha
  • 41. Plants – spreading of RNA silencing short distance long distance
  • 42. Plants –> Animals   ? ? ? environmental & systemic RNAi circulating miRNAs? ? Huang et al., 2006 Baum et al., 2007 Mao et al. 2007 environmental & systemic RNAi
  • 43. Plants –> Animals Unclear/controversial issues: Mechanism of transport • Mechanism of transport across membranes not explained • Unclear if free or bound to a protein • Survival in digestive tract? Effector complex structure • Would require binding of methylated single stranded RNAs by AGO Targeting stoichiometry • Concentrations estimated 68-250 fM – too low • Authors calculate ~850 molecules per cell, cannot be verified – data not released
  • 44. Plants –> Animals • meta-study of xenomiRs of 824 datasets from human tissues and body fluids • xenomiRs commonly present in tissues (17%) and body fluids (69%), • low abundance, 0.001% of host human miRNA counts • no significant enrichment in sequencing data from tissues and body fluids exposed to dietary intake (e.g. liver). • no significant depletion in tissues and body fluids that are relatively separated from the main bloodstream (e.g, brain and cerebro-spinal fluids) • the majority (81%) of body fluid xenomiRs stem from rodents, which are rare human dietary contributions, but common laboratory animals. • body fluid samples from the same studies are clustered by xenomiR compositions - suggesting technical batch effects. • feeding studies - no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. doi: 10.1261/rna.059725.116 RNA, advanced online, Jan 6., 2017
  • 45. Animal Dicer evolution • RNAi-dedicated Dicer-2 in Arthropods is a derived character • the mammalian “miRNA” Dicer is related to miRNA-producing Dicer-1 in Arthropods • Dicer in C. elegans produces efficiently miRNAs and siRNAs Dicer “miRNA” Dicer
  • 46. nucleus cytoplasm GW182 AGO2 AAAAA Cleavage of mRNA by Ago2 Exportin 5-mediated transport AGO AAAAA mRNA degradation Inhibition of translation relocation to P-bodies GW182 miRNA duplex Dicer GW182 AGO RISC loading Mammalian microRNA pathway pri-miRNA pre-miRNA DGCR8 Microprocessor complex DGCR8 Dicer cleavage targeting pre-miRNA Drosha miRISC
  • 47. Mammals (and vertebrates in general) OAS MDA5 TLR3 PACT Dicer miRNA RNAi AGO1-4 gene control AGO1-4 AAAAA inhibition of translation GW182 AGO2 AAAAA mRNA cleavage pre-miRNA miRNA dsRNA siRNA TARBP2 PKR OAS RIG-I translational repression RNAse L IFN signaling interferons & interferon stimulated genes Interferon response common sensors RNA silencing antiviral defense dsRNA • miRNA pathway is the main RNA silencing pathway • main dsRNA response = sequence-independent interferon response
  • 48. Annelids Dicer miRNA RNAi AGO AGO AAAAA inhibition of translation AGO AAAAA mRNA cleavage pre-miRNA miRNA dsRNA siRNA TARBP2 ? OAS RIG-I ? MDA5 ? RNAse L signaling innate immunity? dsRNA response common sensors RNA silencing dsRNA ? ? • almost no functional data, set-up seems similar to mammals
  • 49. Molluscs RdRP Dicer miRNA RNAi AGO gene control & antiviral defense? AGO AAAAA inhibition of translation GW182 AGO AAAAA mRNA cleavage pre-miRNA miRNA dsRNA siRNA TARBP2 PKR OAS RIG-I MDA5 translational repression RNAse L IFN signaling interferons & interferon stimulated genes Interferon response common sensors RNA silencing antiviral defense dsRNA ? MX • almost no functional data, set-up seems similar to mammals • possible RdRP loop – would make it similar to nematodes
  • 50. Arthropods nucleus cytoplasm AGO2 defense gene control AGO2 AAAAA mRNA cleavage siRNA dsRNA PKR RIG-I MDA5 Interferon response common sensors RNAi dsRNA AGO1 gene control AGO1 AAAAA inhibition of translation GW182 pre-miRNA LOQS miRNA miRNA 21-23 nt pri-miRNA Microprocessor R2D2 Dicer-1 Dicer-2 TLR3? signaling innate immunity? • separated miRNA & RNAi • sensors of the interferon response present
  • 51. Interferon response induced by long dsRNA (>30bp) sensing specific responses MDA5 TLR3 PKR OAS RIG-I common response INTERFERON RESPONSE ISG interferon-stimulated genes eIF2a P RNaseL global inhibition of translation global mRNA degradation 2’,5’-OA • The interferon response can be detected/monitored
  • 52. lack of 3’ overhangs induces IFN via Rig-I dsRNA > 30 bp activates PKR and 2’,5’-OAS some sequence motifs within ssRNA can activate IFN cationic lipid-RNA complexes activate IFN via TLR3 and TLR7 5’ triphosphate introduced by phage RNA polymerases activates IFN siRNA < 30 bp can activate PKR lack of 3’ overhangs induces IFN via Rig-I dsRNA > 30 bp activates PKR and 2’,5’-OAS some sequence motifs within ssRNA can activate IFN cationic lipid-RNA complexes activate IFN via TLR3 and TLR7 5’ triphosphate introduced by phage RNA polymerases activates IFN siRNA < 30 bp can activate PKR Interferon response induced by siRNAs 24hours72hours mock siRNA A siRNA B1 siRNA B2 siRNA C mock siRNA A siRNA B1 siRNA B2 siRNA C
  • 53. dsRBDZβ deaminaseZα NLSNES ADAR1p150 ADAR1p110 ADAR2 ADAR3 Adenosine deamination Kono & Akiyama, 2013 DOI: 10.5772/55203 nucleus cytoplasm ADAR1 degradation ?