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The RNAi machinery is involved in chromosome segregation in trypanosomes Philippe BASTIN Muséum National dHistoire Naturelle INSERM U565 & CNRS UMR 5153.

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Présentation au sujet: "The RNAi machinery is involved in chromosome segregation in trypanosomes Philippe BASTIN Muséum National dHistoire Naturelle INSERM U565 & CNRS UMR 5153."— Transcription de la présentation:

1 The RNAi machinery is involved in chromosome segregation in trypanosomes Philippe BASTIN Muséum National dHistoire Naturelle INSERM U565 & CNRS UMR 5153 & MNHN 0503

2 Trypanosoma brucei Flagellated protozoa (sleeping sickness) No RNA pol II promoters Polycistronic transcription Trans-splicing (addition of a splice leader) No introns RNAi (but not in related T. cruzi and Leishmania)

3 Trypanosome gene expression pre-mRNA trans-splicing + poly-adenylation polycistronic transcription genes (no introns) sl splice leader (SL) sl AAA

4 dsRNA mRNA RNAi in trypanosomes mRNA destruction siRNA Fragmentation (DICER activity) RISC mRNA pairing ? Presence of dsRNA

5 T. brucei Argonaute genes TbAGO1 Piwi PAZ TbPWI1 LmPWI1 GlAGO1 SpAGO1 TtTWI1 AtAGO1 CeRDE1 NcQDE2 DmAGO2 polyQ 100 aa RGG Piwi PAZ

6 Cell number K.O. K.O. + GFP::TbAGO1 (-TET) K.O. + GFP::TbAGO1 (+TET) GFP fluorescence Sca I TbAGO1 BSD G418 TbAGO1 Sca I PHLEO TbAGO1GFP Sca I Xba I * Generation of TbAGO1-/- KO cell line KO WT KOKO + GFP::TbAGO1 1 kb KO + GFP::TbAGO1 HYG Tet-repressor

7 WTK.O Cells displaying RNAi phenotype (% population) K.O. + GFP::TbAGO1 (+TET) (-TET) TbAGO1 is required for RNAi Transfection of dsRNA WTKO Northern Blot (PFRA) PHLEO PFRA WTKO dsRNA expression Expression of dsRNA

8 TbAGO1 is required for optimal growth Log cell density (millions/ml) Time (h) W.T. K.O. K.O. + GFP::TbAGO1 (+TET)

9 Phase DAPI Tubulin TbAGO1-/- cells show defects in spindle formation

10 Chromosome segregation in wild-type trypanosomes Tubulin (green) +DAPI (blue) + chromosome VIII (red)

11 Chromosome segregation in wild-type cells Phase+DAPIchromosome IDAPI + FISH

12 Chromosome segregation in AGO1-/- cells

13 TbAGO1-/- shows overexpression of Ingi transcripts RNA Northern blot Ingi Pf16 (loading control) WTKO Shi et al., MCB 2004 Durand-Dubief, Benghanem, Bastin, 2004 Ingi ORF (5.2kb)A TAA B More than 400 copies (mostly truncated) AB Intact Ingi element (LINE family) Intact RIME element (SINE family) Interphase cell Mitotic cell DAPIMiniIngi

14 CONCLUSIONS 1. TbAGO1 is essential for RNAi in T. brucei 2. The RNAi machinery appears required for proper chromosome segregation at mitosis 3. The RNAi machinery is required to control transposons expression 4. RNAi can generate transcriptional gene silencing

15 Muséum National dHistoire Naturelle (Paris) Philippe BASTIN Linda KOHL (CRA CNRS/MC MNHN) Filippo RUSCONI (CR2 CNRS) Mickaël DURAND-DUBIEF (PhD) Sabrina BENGHANEM (PhD) Carole BRANCHE (PhD) Géraldine TOUTIRAIS (ITA, INSERM) Gwénola DORE (MNHN) Sandra NWGABIT (stagiaire) INSERM U565& CNRS UMR5153 & MNHN USM 0503 Carine GIOVANNANGELI & J.S. SUN

16 Muséum National dHistoire Naturelle (Paris) Philippe BASTIN Funding ATIPE CNRS FRM ACI Dynamique et réactivité des assemblages biologiques ACI Biologie du développement GIS (Research on Rare Genetic Diseases) ESF Gouvernement Luxembourgeois EMBO

17 Muséum National dHistoire Naturelle (Paris) Philippe BASTIN Collaborations Derrick ROBINSON (Bordeaux) Mickael BOSHART (Munich) Mark FIELD (London) Frédéric TOURNIER (Paris VII) Bénédicte DURAND (Lyon) Estelle ESCUDIER (Créteil) Serge AMSELEM (Créteil) Jean-François JOANNY (Curie) Thanks to K. GULL, P. ENGLUND, C. CLAYTON, G. CROSS


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