The African trypanosome, Trypanosoma brucei possesses a large and unique intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of 2 major proteins, PFRA and PFRC. We have generated an inducible mutant trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA. When expression of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and reversible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially in new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi approach will have widespread applicability in studies of gene function and cellular processes in parasites.

REFERENCES

Bastin
P.
,
Bagherzadeh
A.
,
Matthews
K. R.
,
Gull
K.
(
1996
).
A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei.
Mol. Biochem. Parasitol
77
,
235
239
Bastin
P.
,
Sherwin
T.
,
Gull
K.
(
1998
).
Paraflagellar rod is vital for trypanosome motility.
Nature
391
,
548
–.
Bastin
P.
,
Pullen
T.
,
Sherwin
T.
,
Gull
K.
(
1999
).
Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl -1.
J. Cell Sci
112
,
3769
3777
Bastin
P.
,
MacRae
T.
,
Francis
S. B.
,
Matthews
K. R.
,
Gull
K.
(
1999
).
Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes.
Mol. Cell. Biol
19
,
8191
8200
Clayton
C. E.
,
Hausler
T.
,
Blattner
J.
(
1995
).
Protein trafficking in kinetoplastid protozoa.
Microbiol. Rev.
59
,
325
344
Cogoni
C.
,
Macino
G.
(
1999
).
Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase.
Nature
399
,
166
169
Cogoni
C.
,
Macino
G.
(
1999
).
Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase.
Science
286
,
2342
2344
Cole
D.
,
Diener
D. R.
,
Himelblau
A. L.
,
Beech
P. L.
,
Fuster
J. C.
,
Rosenbaum
J. L.
(
1998
).
Chlamydomonas kinesin II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons.
J. Cell Biol
141
,
993
1008
Deflorin
J.
,
Rudolf
M.
,
Seebeck
T.
(
1994
).
The major components of the paraflagellar rod of Trypanosoma brucei are two similar but distinct proteins which are encoded by two different gene loci.
J. Biol. Chem
269
,
28745
28751
Eid
J.
,
Sollner-Webb
B.
(
1991
).
Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination.
Proc. Nat. Acad. Sci. USA
88
,
2118
2121
Fire
A.
,
Xu
S.
,
Montgomery
M. K.
,
Kostas
S. A.
,
Driver
S. E.
,
Mello
C. G.
(
1998
).
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature
391
,
806
811
Fire
A.
(
1999
).
RNA-triggered gene silencing.
Trends Genet
15
,
358
363
Gull
K.
(
1999
).
The cytoskeleton of trypanosomatid parasites.
Annu. Rev. Microbiol
53
,
629
655
Hausler
T.
,
Clayton
C. E.
(
1996
).
Post-transcriptional control of hsp70 mRNA in Trypanosoma brucei.
Mol. Biochem. Parasitol
76
,
57
71
Johnson
K. A.
,
Rosenbaum
J. L.
(
1992
).
Polarity of flagellar assembly in Chlamydomonas.
J. Cell Biol
119
,
1605
1611
Ketting
R. F.
,
Haverkamp
T. H. A.
,
van Luenen
H. G. A. M.
,
Plasterk
R. H. A.
(
1999
).
mut -7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RnaseD.
Cell
99
,
133
141
Kohl
L.
,
Sherwin
T.
,
Gull
K.
(
1999
).
Assembly of the paraflagellar rod and of the flagellum attachment zone complex in Trypanosoma brucei.
J. Euk. Microbiol
46
,
105
109
Lee
M. G. S.
,
Van der Ploeg
L. H. T.
(
1990
).
Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei.
Science
250
,
1583
1587
Lee
M. G. S.
,
Van der Ploeg
L. H. T.
(
1997
).
Transcription of protein-coding genes in trypanosomes by RNA polymerase I.
Annu. Rev. Microbiol
51
,
463
489
Lefevbre
P.A.
,
Rosenbaum
J. L.
(
1986
).
Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration.
Annu. Rev. Cell Biol
2
,
517
546
Maga
J. A.
,
Sherwin
T.
,
Francis
S.
,
Gull
K.
,
LeBowitz
J. H.
(
1999
).
Genetic dissection of the Leishmania paraflagellar rod, a unique flagellar cytoskeleton structure.
J. Cell Sci
112
,
2753
2763
Montgomery
M. K.
,
Xu
S. Q.
,
Fire
A.
(
1998
).
RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans.
Proc. Nat. Acad. Sci. USA
95
,
15502
15507
Ngô
H. M.
,
Tschudi
C.
,
Gull
K.
,
Ullu
E.
(
1998
).
Double-stranded RNA induces mRNA degradation in Trypanosomabrucei.
Proc. Nat. Acad. Sci. USA
95
,
14687
14692
Pazour
G. J.
,
Wilkerson
C. G.
,
Witman
G. B.
(
1998
).
A dynein light chain is essential for the retrograde particle movement of intraflagellar transport.
J. Cell Biol
141
,
979
992
Pazour
G. J.
,
Dickert
B. L.
,
Witman
G. B.
(
1999
).
The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly.
J. Cell Biol
144
,
473
481
Porter
M. E.
,
Bower
R.
,
Knott
J.A.
,
Byrd
P.
,
Dentler
W.
(
1999
).
Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas.
Mol. Biol. Cell
10
,
693
712
Rosenbaum
J. L.
,
Cole
D. G.
,
Diener
D. R.
(
1999
).
Intraflagellar transport: the eyes have it.
J. Cell Biol
144
,
385
388
Santrich
C.
,
Moore
L.
,
Bastin
P.
,
Sherwin
T.
,
Brokaw
C.
,
Gull
K.
,
Lebowitz
J. H.
(
1997
).
A motility function for the paraflagellar rod in Leishmania parasites revealed by PFR-2 gene knockouts.
Mol. Biochem. Parasitol
90
,
95
109
Schlaeppi
K.
,
Deflorin
J.
,
Seebeck
T.
(
1989
).
The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical, tandemly linked genes.
J. Cell Biol
109
,
1695
1709
Sherwin
T.
,
Schneider
A.
,
Sasse
R.
,
Seebeck
T.
,
Gull
K.
(
1987
).
Distinct localisation and cell cycle dependence of COOH terminally tyrosinolated-tubulin in the microtubules of Trypanosoma brucei.
J. Cell Biol
104
,
439
446
Sherwin
T.
,
Gull
K.
(
1989
).
The cell division cycle of Trypanosoma brucei brucei: timing of event markers and cytoskeleton modifications.
Phil. Trans. R. Soc. London Ser. B
323
,
575
588
Sherwin
T.
,
Gull
K.
(
1989
).
Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes.
Cell
57
,
211
221
Smardon
A.
,
Spoerke
J. M.
,
Stacey
S. C.
,
Klein
M. E.
,
Mackin
N.
,
Maine
E. M.
(
2000
).
EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans.
Curr. Biol
10
,
169
178
Tabara
H.
,
Sarkissian
S.
,
Kelly
W.J.
,
Fleenor
J.
,
Grishok
A.
,
Timmons
L.
,
Mello
C. C.
,
Fire
A.
(
1999
).
The rde -1 gene, RNA interference, and transposon silencing in C. elegans.
Cell
99
,
123
132
ten Asbroek
A. L. M. A.
,
Ouellette
M.
,
Borst
P.
(
1990
).
Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei.
Nature
348
,
174
175
Tovar
J.
,
Fairlamb
A. H.
(
1996
).
Extrachromosomal, homologous expression of trypanothione reductase and its complementary mRNA in Trypanosoma cruzi.
Nucl. Acids Res
24
,
2942
2949
Vanhamme
L.
,
Pays
E.
(
1995
).
Control of gene expression in trypanosomes.
Microbiol. Rev.
59
,
223
240
Wagner
R. W.
,
Sun
L.
(
1998
).
Double-stranded RNA poses puzzle.
Nature
391
,
744
745
Wirtz
E.
,
Clayton
C. E.
(
1995
).
Inducible gene expression in trypanosomes mediated by a procaryotic repressor.
Science
268
,
1179
1183
Witman
G. B.
(
1975
).
The site of in vivo assembly of flagellar microtubules.
Ann. NY Acad. Sci
253
,
178
191
Woods
A.
,
Sherwin
T.
,
Sasse
R.
,
MacRae
T.H.
,
Baines
A.
,
Gull
K.
(
1989
).
Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.
J. Cell Sci
93
,
491
500
Woodward
R.
,
Gull
K.
(
1990
).
Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei.
J. Cell Sci
95
,
49
57
Zhang
W. W.
,
Matlashewski
G.
(
1997
).
Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2.
Proc. Nat. Acad. Sci. USA
94
,
8807
8811
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