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The RNAi machinery is involved in chromosome segregation in trypanosomes Philippe BASTIN Muséum National d’Histoire Naturelle INSERM U565 & CNRS UMR 5153 & MNHN 0503 [email protected]
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The RNAi machinery is involved in chromosome segregation in
trypanosomes
Philippe BASTIN
Muséum National d’Histoire Naturelle
INSERM U565 & CNRS UMR 5153 & MNHN [email protected]
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)
Trypanosome gene expression
pre-mRNA
trans-splicing + poly-adenylation
polycistronic transcription
genes (no introns)
sl sl sl
sl
splice leader (SL)
sl sl sl sl
AAA AAA AAA
dsRNA
mRNA
RNAi in trypanosomes
mRNA destruction
siRNA
Fragmentation(DICER activity)
RISC
mRNA pairing ?
Presence of dsRNA
T. brucei Argonaute genes
TbAGO1 PiwiPAZ
TbPWI1
LmPWI1
GlAGO1
SpAGO1
TtTWI1
AtAGO1
CeRDE1
NcQDE2
DmAGO2
polyQ
polyQ
100 aa
RGG
PiwiPAZ
Cel
l nu
mb
er
0
200
400
600
800
100
101
102
103
K.O.K.O. + GFP::TbAGO1 (-TET) K.O. + GFP::TbAGO1 (+TET)
GFP fluorescence
Sca I Sca I
TbAGO1
BSD
G418
TbAGO1
Sca I Sca I
PHLEOTbAGO1GFPSca I
Xba I
*
Generation of TbAGO1-/- KO cell line
KO
WT
WT KO KO +GFP::TbAGO1
1 kb
KO + GFP::TbAGO1
HYGTet-repressor
WT K.O.0
10
20
30
40
50
Cel
ls d
isp
layi
ng
RN
Ai
ph
eno
typ
e (
% p
op
ula
tio
n)
K.O. +GFP::TbAGO1(+TET) (-TET)
TbAGO1 is required for RNAi
Transfection of dsRNA
WT KO
Northern Blot (PFRA)
PHLEOPFRAPFRA
WT KO- - + + + +dsRNA
expression
Expression of dsRNA
TbAGO1 is required for optimal growth
1
10
0 6 12 18 24 30
Lo
g c
ell
den
sit
y (m
illio
ns
/ml)
Time (h)
W.T.K.O.K.O. + GFP::TbAGO1 (+TET)
Phase DAPI Tubulin
TbAGO1-/- cells show defects in spindle formation
QuickTime™ et undécompresseur
sont requis pour visionner cette image.
Chromosome segregation in wild-type trypanosomes
Tubulin (green) +DAPI (blue) + chromosome VIII (red)
Chromosome segregation in wild-type cells
Phase+DAPI chromosome I DAPI + FISH
Chromosome segregation in AGO1-/- cells
TbAGO1-/- shows overexpression of Ingi transcripts
RNANorthern blot
Ingi
Pf16 (loading control)
WT KO
Shi et al., MCB 2004Durand-Dubief, Benghanem, Bastin, 2004
Ingi ORF (5.2kb)ATAA
B
More than 400 copies (mostly truncated)
A B
Intact Ingi element (LINE family)
Intact RIME element (SINE family)
Interphase cell
Mitotic cell
DAPI Mini Ingi
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
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 0503Carine GIOVANNANGELI & J.S. SUN
Muséum National d’Histoire Naturelle (Paris)
Philippe BASTIN
FundingATIPE CNRS
FRMACI Dynamique et réactivité des assemblages
biologiquesACI Biologie du développement
GIS (Research on Rare Genetic Diseases)ESF
Gouvernement LuxembourgeoisEMBO
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 toK. GULL, P. ENGLUND, C. CLAYTON, G. CROSS
