The rnai machinery is involved in chromosome segregation in trypanosomes
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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

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The RNAi machinery is involved in chromosome segregation in trypanosomes

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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

[email protected]


Trypanosoma brucei

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

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


The rnai machinery is involved in chromosome segregation in trypanosomes

RNAi in trypanosomes

Presence of dsRNA

dsRNA

Fragmentation

(DICER activity)

siRNA

RISC

mRNA pairing ?

mRNA

mRNA destruction


The rnai machinery is involved in chromosome segregation in trypanosomes

polyQ

polyQ

T. brucei Argonaute genes

RGG

PAZ

Piwi

TbAGO1

TbPWI1

LmPWI1

GlAGO1

SpAGO1

TtTWI1

AtAGO1

CeRDE1

NcQDE2

DmAGO2

PAZ

Piwi

100 aa


The rnai machinery is involved in chromosome segregation in trypanosomes

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


The rnai machinery is involved in chromosome segregation in trypanosomes

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)


The rnai machinery is involved in chromosome segregation in trypanosomes

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)


The rnai machinery is involved in chromosome segregation in trypanosomes

TbAGO1-/- cells show defects in spindle formation

Phase DAPI Tubulin


The rnai machinery is involved in chromosome segregation in trypanosomes

Chromosome segregation in wild-type trypanosomes

Tubulin (green) +DAPI (blue) + chromosome VIII (red)


The rnai machinery is involved in chromosome segregation in trypanosomes

Chromosome segregation in wild-type cells

Phase+DAPI

chromosome I

DAPI + FISH


The rnai machinery is involved in chromosome segregation in trypanosomes

Chromosome segregation in AGO1-/- cells


The rnai machinery is involved in chromosome segregation in trypanosomes

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

DAPIMiniIngi

Ingi

Mitotic cell

Pf16

(loading control)

Shi et al., MCB 2004

Durand-Dubief, Benghanem, Bastin, 2004


The rnai machinery is involved in chromosome segregation in trypanosomes

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


The rnai machinery is involved in chromosome segregation in trypanosomes

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


The rnai machinery is involved in chromosome segregation in trypanosomes

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


The rnai machinery is involved in chromosome segregation in trypanosomes

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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