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Renou JP, Navarro L, Berthomé R, Aubourg S, Taconnat L, Jay F, Bitton F, Elftieh S, Yu A & Voinnet O URGV INRA/CNRS/UEVE, Evry, France IBMP CNRS, Strasbourg.

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Presentation on theme: "Renou JP, Navarro L, Berthomé R, Aubourg S, Taconnat L, Jay F, Bitton F, Elftieh S, Yu A & Voinnet O URGV INRA/CNRS/UEVE, Evry, France IBMP CNRS, Strasbourg."— Presentation transcript:

1 Renou JP, Navarro L, Berthomé R, Aubourg S, Taconnat L, Jay F, Bitton F, Elftieh S, Yu A & Voinnet O URGV INRA/CNRS/UEVE, Evry, France IBMP CNRS, Strasbourg A Genomic Approach to study the pri-MIR & Si regulation in Arabidopsis

2 CATMA is a Eugene-based chip (Crowe et al. NAR, 2003; Hilson et al. Genome Res. 2004) ‏ (Foissac et al. NAR, 2003) ‏ At4g31850 GST have been chosen in specific regions : example of a gene from the PPR/PCMP family with conserved motifs (Aubourg et al. 2000; Small and Peeters 2000) ‏ PPR motif Eugène annotation TAIR annotation GST (amplicons) designed by SPADS (Thareau et al. Bioinformatics, 2003) ‏ A Collection of Gene specific tags (GST) covering all the predicted transcripts (CATMA, coord. P. Hilson), a RNAi resource (AGRIKOLA, coord I. Small) ‏

3 CATMA (V2.1) : GSTs CATMA (V4) : GSTs GFTs = (full genome) ‏ (Sclep et al. BMC Bioinf. 2007) ‏ CATMA (V2.2) : + Chl & Mt probes CATMA (V2.3) : miRNA probes (Navarro et al. in prep) ‏ L. Taconnat, F. Bitton Data analysis: a normalized protocol M.L. Martin-Magniette, coll. OMIP INA-PG (Lurin et al. Plant Cell, 2004) ‏ CATMA advances

4 2003/2007: 130 COLLABORATIONS 19 ANR Programs (GnP & Blan) Many INRA (36), CNRS, CEA and Univ. labs EU Program: (Compedium of Arabidopsis Gene Expression) ‏ Foreign collaborations (Brazil, Singapour, Czech rep., Germany, Belgium, UK, Spain, Sweden) ‏ > 2900 hybridizations A huge data-set covering multiple developmental conditions, biotic and abiotic stresses and mutants 21 publications More to come… (18 in prep or submitted) ‏

5 CATdb: a public access to Arabidopsis transcriptome data from the URGV-CATMA platform (Gagnot et al. NAR 2007, Oct.) ‏ Automatic export and link

6 Mining the data: FLAGdb++,an integrative database coding gene additional annotation tagged mutant GST probes repeat element protein motif Affymetrix probes MPSS tag RNA gene curated annotation gene family/GO Links to… GENBANK – PDB – TIGR - PFAM MIPS – TAIR – ARAMEMNON SWISSPROT – GENEVESTIGATOR eFP BROWSER - CATdb Samson et al. NAR, 2004

7 697 GSTs mapped outside the genes present in the last annotation release (R6.0 from TAIR). 29 GSTs allow to precise gene extremities Analysis of CATMA data and identification of hundreds of novel genes (Aubourg et al BMC Genom.) ‏ GSTs can also be used to detect erroneous gene merging Affymetrix primer set -2,40 2,61 -1,26 0,90 0,60-0,54 Ratios in 3 independent experiments cul3 mutant / wt Nitrogen starv. / control tor mutant / wt 469 GSTs tag novel genes 498 with signal

8 CH 3 TGS Chromatin modification 60S 40S n (A) ‏ Translational inhibition eiF4G RNA silencing core mechanism Sense transgenesEndogenous lociInvert-repeat transgenes Phytoviruses RNA cleavage (A) n PTGS  (A) n Amplification RdRP small RNA duplex 19-25bp (si/miRNA) ‏ RdRP Dicer RISC assembly (AGO) ‏ AGO

9 DCL1: 20-25nt miRNA -Development -many other processes DCL2: 22nt nat-siRNA -viral-derived siRNAs DCL3: 24nt IR-siRNA -DNA methylation -Heretochromatin -TGS DCL4: 21nt siRNA -viral-derived siRNAs -TasiRNAs - ‘Young’ miRNAs Four Dicers in Arabidopsis thaliana >50,000 (200,000) ‏ ~100 (conserved miRNAs) ‏ 2 5 TE

10 Nucl. Cyto. miR/miR* CH3 HEN1 HYL1 RNA Pol II (A) n DCL1 pri-miRNA AGO1 miR (A) n (U) <10 40S 60S AGO1 miR (A) n Degradation Translation inhibition Predominant in animalsPredominant in plants DCL1 pre-miRNA HASTY CH3 The Arabidopsis miRNA pathway

11 How to identify mi/siRNAs precursors transcripts in Arabidopsis on CATMA ? 1217 candidates miRNAs Collaboration : O. Voinnet, L. Navarro (IBMP Strasbourg), B. Arnold (JIT Norwich UK) ‏ 465 non-redundant miR or Si precursors 60 mers oligoprobes added on the CATMA arrays ORF1 ORF2 Bioinformatic analysis: Identification of stable stem- loop structures throughout the Arabidopsis genome miRspot706 = Hunt-729_777 miRspot285 = hunt-556_605 miRspot730 = Hunt-861_908 miRspot327 = hunt-742_790 miRspot1047 = Hunt-193_245 Ex : 5 new precursors validated by Q-PCR (Navarro et al. in prep) ‏

12 hypmiR / miR in FLAGdb++ Query with ID S. Aubourg V. Brunaud F. Samson

13 hypmiR / miR in FLAGdb++ known miR % identity with the closest paralogous region hypmiR S. Aubourg V. Brunaud F. Samson

14 25 miRNA precursors are more elevated in naïve silencing mutant seedlings 22 out of 25 are specifically more elevated in dcl1-9 mutant seedlings 5 miRNA precursors have not yet been identified (yellow) Validation of the microarray approach using silencing mutant seedlings (Navarro et al. in prep) ‏

15 Small RNAs in PAMP-triggered immunity Bacterial cell Yonekura et al., 2003 Flg22 is a “Pathogen associated molecular patterns” that triggers an innate immune response in plants (Felix et al., 1998) ‏ TLR5 FLS2 - Highly conserved - Present in different organisms - Play a pivotal role for the pathogen

16 21 out of 24 flg22-induced pri-miRNA/siRNA are novel 14 are hyper-induced in dcl4 treated seedlings and are likely early evolving pri-miRNAs or siRNA precursors (Navarro et al. in prep) ‏ Small RNAs in PAMP-triggered immunity

17 miRspot730 miRspot292 miRspot300 miRspot495 miRspot1213

18 AuxRE motif ARF TFs RY motif B3-type TFs Novel motif Present in: 14 promoters 13 promoters These cis-acting elements are not over-represented in a promoter set of 29 non Flg22-induced miRNA precursors (Navarro et al. in prep) ‏ Over-representation of regulatory cis-acting elements in Flg22 induced pri-miR promoters

19 Collaborators The ADT members R. Berthomé (CR2), J. Yansouni (IE GnP), S. Balzergue (IE), F. Bitton (IE), S. Mainguet (PhD St), L. Taconnat (AI), A. Yu (IE GnP), S. Elftieh (IE GnP) ‏ S. Pelletier (TR) & D. Glissant (Post-Doc) ‏ The Bioinformatics group A. Lecharny, V. Brunaud, F. Samson & M.L. Martin-Magniette (Stats) ‏


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