10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Purva Tranquillity best living place in East Bangalore
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
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
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
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
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
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
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