Blocking variant surface glycoprotein synthesis in Trypanosoma brucei triggers a general arrest in translation initiation.
BACKGROUND
The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest.


RESULTS
Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1-4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells.


CONCLUSIONS
The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.
Centre for Biomolecular Sciences, University of St. Andrews, Fife, Scotland, United Kingdom.
Terry K
TK
Smith
Nadina
N
Vasileva
Eva
E
Gluenz
Stephen
S
Terry
Neil
N
Portman
Susanne
S
Kramer
Mark
M
Carrington
Shulamit
S
Michaeli
Keith
K
Gull
Gloria
G
Rudenko
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002453:Cell Cycle; D004721:Endoplasmic Reticulum / Q000378:metabolism; D014644:Genetic Variation; D006023:Glycoproteins / Q000737:chemistry; D009696:Nucleic Acids / Q000737:chemistry; D010641:Phenotype; D011132:Polyribosomes / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D034622:RNA Interference; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014353:Trypanosomiasis, African / Q000469:parasitology; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D006023:Glycoproteins; D009696:Nucleic Acids; D014643:Variant Surface Glycoproteins, Trypanosoma