Plant RNAi mechanisms: lessons from silent transgenes · 2015. 8. 4. · Small RNA / AGO association determines the type of silencing 21-22-nt small RNA associate with AGO1, AGO2,
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Plant RNAi mechanisms: �lessons from silent transgenes�
Institut Jean-Pierre Bourgin, INRA Versailles�
miRNA duplex
RNaseIII
RdRP
ssRNA precursor
Pol
folding
siRNA duplexes
dsRNA
Pol
miRNA siRNA population
Argonaute Argonaute
PolPol
RNaseIII RNaseIII RNaseIII
Plants encode two types of small RNAs: miRNA and siRNA�
MIR genes endoIR NAT pairs TAS and PolIV loci�
miRNA duplex
RNaseIII
RdRP
ssRNA precursor
Pol
folding
siRNA duplexes
dsRNA
Pol
miRNA
Artificial RNAi strategies based on endogenous pathways�
siRNA population
Argonaute Argonaute
PolPol
RNaseIII RNaseIII RNaseIII
amiRNA IR-PTGS AS-PTGS S-PTGS�
Different DCL produced small RNA of different sizes�
DCL1 -> 21-nt miRNA (19-25-nt depending on the structure of the stem-loop)�
DCL4 -> 21-nt siRNA�
DCL2 -> 22-nt siRNA�
DCL3 -> 24-nt siRNA �
miRNA precursor
5’ 22nt miRNA 21nt miRNA*
miRNA precursor
… 5’ 21nt miRNA 21nt miRNA* 5’ 5’
miRNA size and precision is not always perfectly controlled�
amiRNA�
expected amiRNA ->
- 24-nt�
- 22-nt�- 21-nt�
Rules for long dsRNA processing by DCLs are not known�
RNAi�24-nt�
22-nt�21-nt�
<- DCL3�
<- DCL2�<- DCL4�
IR1� IR2� IR3�
Num
ber
of
alig
ned
rea
ds
per
mil
lion
0
2000
2000
12000
4000
4000
Small RNA sequencing reveal hot-spots that may be cloning artefacts�
35S:GUS (S-PTGS) �
35S:CHS (cosuppression) �
Small RNA / AGO association determines the type of silencing�
21-22-nt small RNA associate with AGO1, AGO2, AGO7 and AGO10. �
If they are homologous to transcribed regions, they guide RNA cleavage or
translational repression.�
If they are homologous to promoter regions, they have no known effect.�
24-nt small RNA associate with AGO4, AGO6 or AGO9.�
If they are homologous to promoter regions, they guide RNA directed DNA
methylation (RdDM), which causes TGS. �
If they are homologous to transcribed regions, they guide DNA methylation
of gene body, which has no consequence on transcription or RNA stability.�
24-nt siRNA/RdDM/TGS is a complex pathway, which regulates �
5000+ endogenous loci (mostly transposons and intergenic repeats)�
DDM1
MET1
HDA6
PolIV
AGO4
DCL3
24-nt siRNA
RDR2
HEN1
CLSY1 PolV
RNA
CLSY1
DRD1
CMT3
DRM2
SUVH
dsRNA
DNA
CMT2
initiation
maintenance
SHH1
Engineering TGS/RdDM is not obvious�
dsRNA producing 35S siRNA are very efficient against 35S-driven transgenes Silencing is inherited after elimination of dsRNA Time to re-expression depends on CG density
dsRNA producing siRNA against endogenous promoters are not efficient Rapid re-expression after elimination of dsRNA
Tethering of SUVH2/9 to target promoter helps triggering RdDM
21-nt small RNAs guide target RNA cleavage�
Mismatches on one side (5’ of the miRNA) are disruptive�
small RNA/target RNA pairs tolerate mismatches and large bulges�
21-nt small RNAs also guide translational repression �
- AGO1
- RbcS0.00.51.01.5
mut
ant/c
ontro
l A
GO
1 m
RN
A
7.6 1.0 9.4
Rules for small RNA-mediated translational repression are not known�
--> Whether small RNA affect translation of unexpected targets cannot be predicted�
21-nt guide RNA cleavage and degradation�
target mRNA 5’
PAZ
PIWI
Mid 3’ 5’
AGO1
3’
5’
XRN EXO
degradation
21ntsRNA
RNA cleavage
Small RNA size determines the outcome of target RNAs :�
3’
3’
5’ dsRNA
DRB4
Population of 21-nt siRNA duplex
3’
cleaved RNAs
5’
3’ 3’ 3’ 3’
SGS3 RDR6
HEN1
DCL4
target mRNA 5’
PAZ
PIWI
Mid 3’ 5’
AGO1 22nt
sRNA
RNA cleavage
Small RNA size determines the outcome of target RNAs :�
22-nt guide RNA cleavage and production of secondary 21-nt�
target mRNA 5’
PAZ
PIWI
Mid 3’ 5’
AGO1
3’
5’
XRN EXO
degradation
3’
3’
5’ dsRNA
DRB4
Population of 21-nt siRNA duplex
3’
cleaved RNAs
5’
21ntsRNA
RNA cleavage
3’ 3’ 3’ 3’
SGS3 RDR6
HEN1
DCL4
target mRNA 5’
PAZ
PIWI
Mid 3’ 5’
AGO1 22nt
sRNA
RNA cleavage
22-nt guide RNA cleavage and production of secondary 21-nt�
Small RNA size determines the outcome of target RNAs :�
dcl1 dcl1 dcl3 dcl1 dcl4 dcl1 dcl3 dcl4
What happens in the absence of DCL1 and DCL4 ?�
dcl1 dcl2 dcl1 dcl2 dcl3
dcl1 dcl1 dcl3 dcl1 dcl4 dcl1 dcl3 dcl4
dcl1 dcl2 dcl4 dcl1 dcl2 dcl3 dcl4
DCL2 has deleterious effect in the absence of DCL1 and DCL4�
In dcl1 dcl4, which lacks 21-nt siRNAs, 22-nt siRNAs made by DCL2 promote
secondary 22-nt siRNAs, which promote tertiary 22-nt siRNAs, which promote…�
dcl1 dcl4 produce a cascade of 22-nt �
3’
3’
5’ dsRNA
DRB4
Population of 22-nt siRNA duplex
3’
cleaved RNAs
5’
3’ 3’ 3’ 3’
SGS3 RDR6
HEN1
DCL2
target mRNA 5’
PAZ
PIWI
Mid 3’ 5’
AGO1 22nt
sRNA
RNA cleavage3’
3’
5’ dsRNA
DRB4
Population of 22-nt siRNA duplex
3’
cleaved RNAs
5’
3’ 3’ 3’ 3’
SGS3 RDR6
HEN1
DCL2
target mRNA 5’
PAZ
PIWI
Mid 3’ 5’
AGO1 22nt
sRNA
RNA cleavage
-> will this amiRNA trigger the production of secondary siRNAs ?�
24-nt�
22-nt�21-nt�
The amount of 22-nt necessary to trigger the production of
secondary siRNAs is not known�
amiRNA�
-> will these secondary siRNAs have off-target effects ?�
Progression of silencing�
PTGS involves non cell autonomous siRNA�
PTGS is initiated locally and then spreads systemically�
PTGS produces a sequence-specific systemic silencing signal�
apex�grafting�
apex�grafting� Homologous
transgenes�
Non-�homologous transgenes�
NS scion� PTGS stock� PTGS scion�
NS scion� PTGS stock� NS scion�
Unlike siRNAs, miRNAs (and artificial miRNAs) �
mostly act in a cell autonomous manner�
Why siRNAs, but not miRNAs, move from cell to cell is not known�
Within siRNA populations, movement is not homogenous�
WT mutant
Mock CMV Mock CMV
PTGS-deficient mutants are hyper-susceptible to viruses
WT rdr6 sgs3
CMV-CP�
Role of the RNAi machinary in distinguishing self from non-self�
Viral RNA
Virus
Virus
replication
dsRNA
Antiviral PTGS model
�������
���������������
siRNA 21-22nt
Virus
Initiation
dsRNA
Viral RNA
Virus
replication
Antiviral PTGS model
������
���������������
siRNA
Virus
Antiviral PTGS model
21-22nt
�������
�����
�����
Viral RNA
Amplification
AGO1/2
dsRNA
�����
Role of the RNAi machinary in distinguishing self from non-self�
What is outcome of ectopic DNA and RNA during:�
- Duplication�
- Transposition�
- Transformation�
Transposon-mediated TGS�
Col Ler Ws Kas C24 Ita Cvi
Duplication-mediated TGS�
Duplication-mediated PTGS�
Petunia « red-star »CHS duplication
Transgenic Petunia 35S::CHS
Duplication-mediated PTGS�
Petunia « red-star »CHS duplication
Transgenic Petunia 35S::CHS
The H3K4me2/3 demethylase JMJ14 is required for PTGS
jmj14 reduces transgene transcription
polII occupancy�
L1 L1/jmj14 2a3 2a3/jmj14 JAP3 JAP3/jmj14 S-PTGS S-PTGS IR-PTGS
gDNA +RT -RT 35S:NIA2 pre-mRNA EF1∝
GUS
25S
jmj14 also reduces the transcription of non-silenced transgenes�
0�
0,5�
1�
1,5�
35S� GUS5'� GUS3'�
Fol
d C
hang
e�
6b4� 6b4/jmj14-4�
0�
0,5�
1�
1,5�
35S� GUS5'� GUS3'�
Fol
d C
hang
e (H
3K4m
e3)�
6b4� 6b4/jmj14-4�
polII occupancy�H3K4me3 level�
� JMJ14 promotes high levels of transgene transcription,�
which are required but not sufficient for PTGS
In some lines, PTGS affects only a fraction of the population, �
at each generation�
20% �PTGS�
80% �NS�
20% �PTGS�
80% �NS�
20% �PTGS�
80% �NS�
Hc1�
40% �PTGS�
60% �NS�
40% �PTGS�
60% �NS�
40% �PTGS�
60% �NS�
Hc2�
Could PTGS frequency depend on the probably that a transgene locus
produce aberrant RNA above the threshold level that RNA quality
control (RQC) pathways can handle ?�
0
10
20
30
40
50
60
70
80
90
100
H
Per
cent
age
of s
ilenc
ed p
lant
s
RQC counteracts PTGS �
Hc1� Hc1�xrn2�
Hc1�xrn3�
Hc1�xrn4�
Hc1�rrp4�
Hc1�rrp41�
Hc1�rrp44�
Hc1�rrp6l1�
3’->5’ exoribonuclease activity: RRP4, RRP6L1, RRP41, RRP44 �
5’->3’ exoribonuclease activity : XRN2, XRN3, XRN4, FRY1�
P-body decapping components: DCP1, VCS�
Hc1�fry1�
Hc1�dcp1�
Hc1�vcs�
virus / transgenetransgeneaberrant RNA
Low levels of transgene aberrant RNA are degraded by RQC�
EXO XRN
mRNA
virus / transgene
dsRNA
AGO 1
AGO 1
SDE5 SGS3
RDR6
DRB4HEN1
transgene
Amplification�
DCL4
THO/TREX
siRNA
DCL2
High levels of transgene aberrant RNA saturate RQC�
Initiation�
EXO XRN
aberrant RNA mRNA
RFP:DCP1 X GFP:SGS3
P-bodies and siRNA bodies are distinct but adjacent�
CFP:DCP1 X GFP:SGS3
CFP:DCP1
GFP:SGS3
merge
10 μm
5 μm
GFP
CFP
Collaboration M. Crespi (CNRS, Gif) and A. Maizel (Heidelberg Univ)�
Who’s doing the work?
Nathalie Bouteiller�Nicolas Butel�Taline Elmayan�Ivan Le Masson�Hervé Vaucheret�Agnès Yu�
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