Plant RNAi mechanisms: lessons from silent transgenes Institut Jean-Pierre Bourgin, INRA Versailles
Plants encode two types of small RNAs: mirna and sirna MIR genes endoir NAT pairs TAS and PolIV loci Pol Pol Pol Pol ssrna precursor folding RdRP dsrna RNaseIII RNaseIII RNaseIII RNaseIII mirna duplex sirna duplexes mirna sirna population Argonaute Argonaute
Artificial RNAi strategies based on endogenous pathways amirna IR-PTGS AS-PTGS S-PTGS Pol Pol Pol Pol ssrna precursor folding RdRP dsrna RNaseIII RNaseIII RNaseIII RNaseIII mirna duplex sirna duplexes mirna sirna population Argonaute Argonaute
Different DCL produced small RNA of different sizes DCL1 -> 21-nt mirna (19-25-nt depending on the structure of the stem-loop) mirna precursor mirna precursor 5 5 21nt mirna 22nt mirna 5 21nt mirna* 5 21nt mirna* DCL4 -> 21-nt sirna DCL2 -> 22-nt sirna DCL3 -> 24-nt sirna
mirna size and precision is not always perfectly controlled amirna - 24-nt expected amirna -> - 22-nt - 21-nt
Rules for long dsrna processing by DCLs are not known IR1 IR2 IR3 RNAi 24-nt 22-nt 21-nt <- DCL3 <- DCL2 <- DCL4
Small RNA sequencing reveal hot-spots that may be cloning artefacts 12000 Number of aligned reads per million 4000 2000 0 2000 4000 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 HDA6 maintenance CMT2 DNA PolIV SHH1 MET1 CLSY1 SUVH CMT3 DRM2 DRD1 dsrna RDR2 DCL3 initiation CLSY1 PolV AGO4 RNA HEN1 24-nt sirna
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 mutant/control AGO1 mrna 1.5 1.0 0.5 0.0 7.6 1.0 9.4 - AGO1 - RbcS Rules for small RNA-mediated translational repression are not known --> Whether small RNA affect translation of unexpected targets cannot be predicted
Small RNA size determines the outcome of target RNAs : 21-nt guide RNA cleavage and degradation AGO1 21nt srna Mid PAZ 5 PIWI 5 target mrna RNA cleavage 5 EXO XRN degradation
Small RNA size determines the outcome of target RNAs : 22-nt guide RNA cleavage and production of secondary 21-nt AGO1 22nt srna Mid PAZ 5 PIWI 5 target mrna RNA cleavage 5 SGS3 RDR6 cleaved RNAs DCL4 DRB4 HEN1 5 dsrna Population of 21-nt sirna duplex
Small RNA size determines the outcome of target RNAs : 22-nt guide RNA cleavage and production of secondary 21-nt AGO1 21nt srna Mid PAZ 5 PIWI 5 target mrna AGO1 22nt srna Mid PAZ 5 PIWI 5 target mrna RNA cleavage EXO XRN degradation 5 RNA cleavage SGS3 RDR6 cleaved RNAs 5 DCL4 DRB4 HEN1 5 dsrna Population of 21-nt sirna duplex
What happens in the absence of DCL1 and DCL4? dcl1 dcl1 dcl3 dcl1 dcl4 dcl1 dcl3 dcl4
DCL2 has deleterious effect in the absence of DCL1 and DCL4 dcl1 dcl1 dcl3 dcl1 dcl4 dcl1 dcl3 dcl4 dcl1 dcl2 dcl1 dcl2 dcl3 dcl1 dcl2 dcl4 dcl1 dcl2 dcl3 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 AGO1 22nt srna Mid PAZ 5 5 target mrna AGO1 22nt srna Mid PAZ 5 5 target mrna PIWI PIWI RNA cleavage 5 RNA cleavage 5 SGS3 RDR6 cleaved RNAs SGS3 RDR6 cleaved RNAs 5 dsrna 5 dsrna DCL2 DRB4 HEN1 Population of 22-nt sirna duplex DCL2 DRB4 HEN1 Population of 22-nt sirna duplex
The amount of 22-nt necessary to trigger the production of secondary sirnas is not known amirna 24-nt 22-nt 21-nt -> will this amirna trigger the production of secondary sirnas? -> will these secondary sirnas have off-target effects?
PTGS involves non cell autonomous sirna PTGS is initiated locally and then spreads systemically Progression of silencing
PTGS produces a sequence-specific systemic silencing signal apex grafting Homologous transgenes NS scion PTGS stock PTGS scion apex grafting Non- homologous transgenes 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
Role of the RNAi machinary in distinguishing self from non-self PTGS-deficient mutants are hyper-susceptible to viruses WT mutant Mock CMV Mock CMV WT rdr6 sgs3 CMV-CP
Antiviral PTGS model Virus Viral RNA Virus replication dsrna
Antiviral PTGS model Virus Viral RNA Virus replication Initiation dsrna sirna 21-22nt
Antiviral PTGS model Virus AGO1/2 Viral RNA Amplification dsrna sirna 21-22nt
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 L1 L1/jmj14 2a3 2a3/jmj14 JAP3 JAP3/jmj14 S-PTGS S-PTGS IR-PTGS jmj14 reduces transgene transcription polii occupancy gdna +RT -RT 35S:NIA2 pre-mrna EF1
jmj14 also reduces the transcription of non-silenced transgenes Fold Change 1,5 1 0,5 0 polii occupancy 35S GUS5' GUS3' GUS 25S Fold Change (H3K4me3) 1,5 1 0,5 0 H3K4me3 level 35S GUS5' GUS3' 6b4 6b4/jmj14-4 6b4 6b4/jmj14-4 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 Hc1 Hc2 20% PTGS 80% NS 40% PTGS 60% NS 20% PTGS 80% NS 20% PTGS 80% NS 40% PTGS 60% NS 40% PTGS 60% NS 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?
RQC counteracts PTGS ->5 exoribonuclease activity: RRP4, RRP6L1, RRP41, RRP44 5 -> exoribonuclease activity : XRN2, XRN3, XRN4, FRY1 P-body decapping components: DCP1, VCS 100 90 Percentage of silenced plants 80 70 60 50 40 30 20 10 0 H Hc1 Hc1 rrp4 Hc1 Hc1 rrp6l1 rrp41 Hc1 rrp44 Hc1 xrn2 Hc1 xrn3 Hc1 xrn4 Hc1 fry1 Hc1 dcp1 Hc1 vcs
Low levels of transgene aberrant RNA are degraded by RQC aberrant RNA virus transgene / mrna XRN EXO
High levels of transgene aberrant RNA saturate RQC aberrant RNA virus transgene / THO/TREX mrna XRN Initiation EXO AGO 1 AGO 1 SGS3 RDR6 SDE5 Amplification dsrna DCL2 DRB4 DCL4 HEN1 sirna
P-bodies and sirna bodies are distinct but adjacent CFP:DCP1 X GFP:SGS3 RFP:DCP1 X GFP:SGS3 5 μm merge CFP:DCP1 GFP CFP 10 μm GFP:SGS3 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