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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]
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
genes (no introns)
splice leader (SL)
polycistronic transcription
sl
sl
sl
sl
pre-mRNA
sl
trans-splicing + poly-adenylation
sl
sl
AAA
sl
AAA
AAA
RNAi in trypanosomes
Presence of dsRNA
dsRNA
Fragmentation
(DICER activity)
siRNA
RISC
mRNA
mRNA pairing ?
mRNA destruction
T. brucei Argonaute genes
RGG
PAZ
TbAGO1
Piwi
TbPWI1
LmPWI1
GlAGO1
SpAGO1
TtTWI1
AtAGO1 polyQ
CeRDE1
NcQDE2
DmAGO2
polyQ
100 aa
PAZ
Piwi
Generation of TbAGO1-/- KO cell line
WT
Sca I
Sca I
TbAGO1
Sca I
Sca I
K.O.
TbAGO1
WT
KO
KO +
K.O. +
GFP::TbAGO1
(-TET)
GFP::TbAGO1
K.O. + GFP::TbAGO1 (+TET)
1 kb
800
Cell number
KO
BSD
600
400
G418
200
0
KO + GFP::TbAGO1
10
0
10
1
10
2
GFP fluorescence
Xba I
*
GFP
Sca I
PHLEO
TbAGO1
Tet-repressor
HYG
10
3
TbAGO1 is required for RNAi
Cells displaying RNAi phenotype
(% population)
Transfection of dsRNA
Expression of dsRNA
PFRA
PFRA
PHLEO
50
dsRNA
expression
40
WT KO
-
WT
+ +
KO
+
+
30
20
10
0
WT
K.O.
K.O. +
GFP::TbAGO1
(+TET) (-TET)
Northern Blot (PFRA)
Log cell density (millions/ml)
TbAGO1 is required for optimal growth
10
W.T.
K.O.
K.O. + GFP::TbAGO1 (+TET)
1
0
6
12
18
Time (h)
24
30
TbAGO1-/- cells show defects in spindle formation
Phase
DAPI
Tubulin
Chromosome segregation in wild-type
trypanosomes
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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
RNA
Northern blot
WT
Intact RIME element
(SINE family)
Intact Ingi element
(LINE family)
KO
TAA
A
Ingi ORF (5.2kb)
B
A 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
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 0503
Carine GIOVANNANGELI & J.S. SUN
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
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