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

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siRNA 21-22nt

Virus

Initiation

dsRNA

Viral RNA

Virus

replication

Antiviral PTGS model

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siRNA

Virus

Antiviral PTGS model

21-22nt

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Viral RNA

Amplification

AGO1/2

dsRNA

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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�