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The RNAi machinery is involved in chromosome segregation in trypanosomes

The RNAi machinery is involved in chromosome segregation in trypanosomes. Philippe BASTIN Muséum National d’Histoire Naturelle INSERM U565 & CNRS UMR 5153 & MNHN 0503 pbastin@mnhn.fr. Trypanosoma brucei. Flagellated protozoa (sleeping sickness) No RNA pol II promoters

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The RNAi machinery is involved in chromosome segregation in trypanosomes

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  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 pbastin@mnhn.fr

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

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

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

  6. Sca I Sca I Sca I Sca I TbAGO1 TbAGO1 BSD K.O. K.O. + GFP::TbAGO1 (-TET) K.O. + GFP::TbAGO1 (+TET) G418 800 Xba I Sca I * GFP TbAGO1 600 PHLEO Cell number 400 200 0 GFP fluorescence WT KO KO + GFP::TbAGO1 0 3 2 1 10 10 10 10 Generation of TbAGO1-/- KO cell line WT 1 kb KO KO + GFP::TbAGO1 Tet-repressor HYG

  7. PFRA PFRA PHLEO WT KO WT KO dsRNA expression - - + + + + 50 40 30 Northern Blot (PFRA) 20 10 0 TbAGO1 is required for RNAi Transfection of dsRNA Expression of dsRNA Cells displaying RNAi phenotype (% population) WT K.O. K.O. + GFP::TbAGO1 (-TET) (+TET)

  8. 10 1 0 6 12 18 24 30 TbAGO1 is required for optimal growth W.T. K.O. K.O. + GFP::TbAGO1 (+TET) Log cell density (millions/ml) 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 Intact RIME element (SINE family) Intact Ingi element (LINE family) Northern blot TAA WT KO A B A Ingi ORF (5.2kb) B More than 400 copies (mostly truncated) Interphase cell DAPI Mini Ingi Ingi Mitotic cell Pf16 (loading control) 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

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