Philippe BASTIN Muséum National d’Histoire Naturelle

Présentation au sujet: "Philippe BASTIN Muséum National d’Histoire Naturelle"— Transcription de la présentation:

1 The RNAi machinery is involved in chromosome segregation in trypanosomes
Philippe BASTIN Muséum National d’Histoire 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
genes (no introns) splice leader (SL) polycistronic transcription pre-mRNA sl sl sl sl sl trans-splicing + poly-adenylation sl sl sl AAA AAA AAA

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

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

6 Generation of TbAGO1-/- KO cell line
WT TbAGO1 Sca I Sca I TbAGO1 Cell number 200 400 600 800 10 1 2 3 K.O. K.O. + GFP::TbAGO1 (-TET) K.O. + GFP::TbAGO1 (+TET) GFP fluorescence WT KO KO + GFP::TbAGO1 1 kb KO BSD G418 KO + GFP::TbAGO1 PHLEO TbAGO1 GFP Sca I Xba I * Tet-repressor HYG

7 Cells displaying RNAi phenotype
TbAGO1 is required for RNAi Transfection of dsRNA Expression of dsRNA 10 20 30 40 50 WT KO Northern Blot (PFRA) PHLEO PFRA dsRNA expression Cells displaying RNAi phenotype (% population) WT K.O. K.O. + GFP::TbAGO1 (+TET) (-TET)

8 TbAGO1 is required for optimal growth
K.O. K.O. + GFP::TbAGO1 (+TET) 1 10 Log cell density (millions/ml) 6 12 18 24 30 Time (h)

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

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

11 Chromosome segregation in wild-type cells
Phase+DAPI chromosome I DAPI + FISH

12 Chromosome segregation in AGO1-/- cells

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

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

15 Muséum National d’Histoire 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 d’Histoire 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 d’Histoire 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