Journée de la Transcriptomique Végétale URGV-Génopôle
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1 Journée de la Transcriptomique Végétale URGV-Génopôle Identification of PPR protein RNA substrates using omic approaches Hakim Mireau, INRA-IJPB Versailles
2 The different genetic compartments of plant cells 1.7 to 2 billion years 1 to 1.5 billion years (FromTimmiset al., 2004 )
3 Plant mitochondrial genome coding capacity From 200 kpb to kpb Arabidopsis (367 kpb) Arabidopsis thaliana 60 genes on 10% of the genome 33 protein coding genes 3 rrna 21 trna 60% of genome with no function or known origin 22 pairs of repeats of more than 100pb, with two major ones of 6,5 and 4,2kb 74 ORFs > 300 nt with no known function (= 10% of the genome) (Unseld et al., 1997)
4 PPR and gene expression in organelles Transcription (multiple promoters) PPR Editing PPR (>400 sites) U U U U U UU intron PPR Splicing (23 group I and II introns) promoter C C C C C CC exon A exon B C C C C C CC intron IR DNA Pre-mRNA Processing PPR Stabilisation PPR mature RNA Translation (no SD) PPR Protein
5 Motif structure of PPR proteins PPR-P (Pure) PPR-PLS E subclass DYW subclass PPR repeats C-terminal domains (Small, 2000; Schmitz-Linneweber, 2008)
6 PPR and gene expression in organelles Transcription (multiple promoters) PPR promoter C C C C C CC exon A exon B C C C C C CC intron IR DNA Editing PPR (>400 sites) U U U U U UU intron PPR Splicing (23 group I and II introns) Processing PPR Stabilisation PPR Translation (no SD) PPR
7 Identification of the RNA target(s) of an RBP 1 - The RIP-Chip (RNA Immunoprecipitation - Chip) approach 2 - The RIP-Seq (RNA Immunoprecipitation RNA seq) approach Target RNA Specific antibody RNA binding protein (PPR)
8 Identification of the RNA target(s) of an RBP 1 - The RIP-Chip (RNA Immunoprecipitation - Chip) approach 2 - The RIP-Seq (RNA Immunoprecipitation RNA seq) approach Target RNA anti-tag antibody RNA binding protein (PPR)
9 Identification of the RNA target(s) of an RBP IP Supernatant IP Pellet
10 Identification of the RNA target(s) of an RBP 1st step : The immunoprecipitation Contaminating RNA Target RNA Wash! - Extraction under conditions maintaining complex integrity. - Use of cross-linking reagents (formaldehyde, UV)? - Defining volume of antibody to use to get maximum enrichment. - Washing conditions to be defined.
11 Identification of the RNA target(s) of an RBP 1st step : The immunoprecipitation Contaminating RNA Target RNA - Extraction under conditions maintaining complex integrity. - Use of cross-linking reagents (formaldehyde, UV)? - Defining volume of antibody to use to get maximum enrichment. - Washing conditions to be defined.
12 Identification of the RNA target(s) of an RBP 1st step : The immunoprecipitation Contaminating RNA Contaminating RNA = Target RNA Target RNA vs Wild-type and proper antibody
13 Identification of the RNA target(s) of an RBP 1st step : The immunoprecipitation Contaminating RNA Contaminating RNA = Target RNA Target RNA vs Wild-type and proper antibody vs Mutant
14 Identification of the RNA target(s) of an RBP 1st step : The immunoprecipitation Contaminating RNA Contaminating RNA = Target RNA Target RNA vs Wild-type and proper antibody vs different antibody
15 Identification of the RNA target(s) of an RBP 1st step : The immunoprecipitation Contaminating RNA Contaminating RNA = Target RNA Target RNA vs Wild-type and proper antibody vs different antibody
16 Identification of the RNA target(s) of an RBP Contaminating RNA Target RNA IP Supernatant Contaminating RNA Target RNA IP Pellet
17 Identification of the RNA target(s) of an RBP 2nd step : (Specific) elution of co-enriched RNAs RNA binding protein Target RNA(s) Contaminating RNA (rrna!) - Elution conditions are important (specific or SDS). - Preparation of co-enriched RNA (phenol extraction) - Reverse cross-linking if necessary - Depletion of rrna (poly A or RiboMinus TM ) - Direct labelling (Micromax ASAP RNA labeling kit, Perking Elmer) or amplification.
18 Identification of the RNA target(s) of an RBP 2nd step : Quantitative analysis of co-enriched RNAs vs Target RNA(s) Contaminating RNA (rrna!) Target RNA(s) Contaminating RNA (rrna!) Wild-type and proper antibody vs Mutant or different antibody
19 Identification of the RNA target(s) of an RBP 2nd step : Quantitative analysis of co-enriched RNAs Chip hybridization Target RNA(s) Contaminating RNA (rrna!) RNA seq
20 Identification of the RNA target(s) of an RBP IP WT and proper antibody IP Mutant or different antibody vs
21 The Arabidopsis PPR336 Mitochondrial Presequence S P P P P P P S -One of the most expressed PPR 6P + 2S motifs 408 aa (45 kda) - Mitochondrially-targeted, membrane-bound - RNA binding protein - Part of a mitochondrial complex BN PAGE I+III 2 CI (kda) CV CIII CIV Respiratory complexes PPR336 Uyttewaal et al, 2008
22 PPR336 is part of an RNA-containing HMW complex 2000 Blue-Native PAGE PPR336 -RNase +RNase -RNase +RNase Uyttewaal et al, 2008
23 PPR336 is associated to mitochondrial polysomes PPR336 HAUT Contrôle BAS traitement Puromycine HAUT BAS RPS1 40 NAD9 24 ARNr 5S PPR336 traitement EDTA HAUT BAS HAUT traitement RNAse BAS RPS1 40 NAD9 24 ARNr 5S Uyttewaal et al, 2008
24 Analysis of RNA co-enriched with PPR336 (RIP-Chip) Anti-PPR336 Anti-NAD9 IP fraction (pellet) Supernatant (S) 1/20 IP fraction (pellet) Supernatant (S) 1/20 RNA extraction (phenol) RNA extraction (phenol) Labeling Labeling (Cy5) (635 nm) (Cy3) (532 nm) (Cy5) (635 nm) (Cy3) (532 nm) Hybridization Hybridization Hybridization to CATMA2.2 (with probes covering most mitochondrial genes ) Relative enrichment E = log2 ((F IP -B IP )/(F S -B S )) Differential enrichment E336-ENAD9
25 8 Enrichissement différentiel (E 336 -E 3HA ) Analysis of RNA co-enriched with PPR336 (RIP-Chip) Valeurs des signaux (F532- B532) RPS3-g MITO349 MITO329 MITO312 MITO294 MITO275 MITO260 MITO248 MITO235 MITO222 MITO206 MITO194 MITO180 MITO164 MITO149 MITO125 MITO109 MITO093 MITO076 MITO058 MITO045 MITO026 MITO011 F532 - B532 (IP 336 No trna) F532 Median - B532 (E 336 -E NAD9 ) Experiment 1 (F 532 -B 532 ) IP336 Surpernatant log (IP/S )336 / (IP/S) NAD9 NotRNA E336-ENAD9 trna (E 336 -E NAD9 ) Experiment 2 (F 532 -B 532 ) IPNAD9 Supernatant
26 RNA co-enriched with PPR336 (%) séquences codantes Coding sequences séquences codantes Coding + intron sequences + 5 UTR séquences codantes + 5'UTR intron 5'UTR séquences intergéniques Intergenic 5 UTR Introns 3 UTR sequences Multiple mitochondrial mrna were identified, and the vast majority corresponded to coding sequences. PPR336 behaves like a RNA binding protein with little specificity.
27 Molecular analysis of the ppr22 mutant Search for c-type cytochrome deficiencies in the mutant Detection of cytochrome c and c1 by western blot analysis (cytochrome c1 is constitutive of complex III and cytochrome c navigates between complex III and IV) No cytochromes c and c1 can be detected in the mutant J. Dahan unpublished
28 Biochemical analysis of ppr22 mutant Cytochromes c/c1 maturation CcmF complex Heme lyase Hamel et al., 2009 Detection of heme lyase subunits ONLY CcmF N1,F N2 et F C are encoded in mitochondria The CcmFN1 and CcmFN2 proteins seem to accumulate normally (their corresponding mrnas are therefore nicely translated) RNA target of R22? A mitochondrial gene of unknown function? J. Dahan unpublished
29 Functional complementation of the ppr22 mutant Col-0 Ho ppr22 plant pppr22-ppr22cds-ha 5 weeks after germination J. Dahan unpublished
30 Rip-Seq on PPR22, workflow Col.0 P R22 -R22-HA Mitochondria purification Mitochondria purification IP Col.0 (anti-ha AB) 3 repeats IP P R22 -R22-HA (anti-ha AB) 3 repeats Pellet Col Supernatant Col Pellet R22 Supernatant R22 - Elution made with HA peptide. -No RiboMinus TM performed. - Libraries have been prepared (no polya selection, light fragmentation, RT). -Sequencing is in progress (Illumina 100bp paired-end, multiplexing /sample).
31 Rip-Seq on PPR22, quality of extracted RNA Col0 Pellet 2 (13 ng/µl) PR22HA Pellet 2 (14 ng/µl)
32 Rip-Seq on PPR22, workflow Col.0 P R22 -R22-HA Mitochondria purification Mitochondria purification IP Col.0 (anti-ha AB) 3 repeats IP P R22 -R22-HA (anti-ha AB) 3 repeats Pellet Col Supernatant Col Pellet R22 Supernatant R22 - Elution made with HA peptide. -No RiboMinus TM performed. - Libraries have been prepared (no polya selection, light fragmentation, RT). -Sequencing is in progress (Illumina 100bp paired-end, multiplexing bp/sample).
33 Rip-Seq on PPR22, data processing To be defined in details but : -«Informatic» elimination rrna -Identification of most represented specificaly-enriched mrna species in the IP R22 sample. - Statistical analysis (ML. Martin-Magnette, URGV).
34 N. ARNAL M. QUADRADO M. UYTTEWAAL (PPR336) J. DAHAN (PPR22) N. VRIELYNCK H. MIREAU F. BUDAR Thanks! The Transcriptomic plateform S. Balzergue, E. Delanoy, C. Lurin, Yansouni J, Renou JP, ML Martin- Magniette. Institut Jean-Pierre Bourgin
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