Blocking variant surface Glycoprotein synthesis in Trypanosoma brucei triggers a general arrest in translation initiation

Smith, T. K. , Vasileva, N. , Gluenz, E. , Terry, S. , Portman, N. , Kramer, S. , Carrington, M. , Michaeli, S. , Gull, K. and Rudenko, G. (2009) Blocking variant surface Glycoprotein synthesis in Trypanosoma brucei triggers a general arrest in translation initiation. PLoS ONE, 4(10), e7532. (doi: 10.1371/journal.pone.0007532) (PMID:19855834) (PMCID:PMC2762041)

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Abstract

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. Methodology/Principal Findings 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.

Item Type:Articles
Additional Information:This research was funded by the Wellcome Trust. T.K.S. and G.R. are Wellcome Trust Senior Research Fellows in the Basic Biomedical Sciences. K.G. is a Wellcome Trust Principal Research Fellow. Work in the lab of M.C. is funded by the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gluenz, Dr Eva
Authors: Smith, T. K., Vasileva, N., Gluenz, E., Terry, S., Portman, N., Kramer, S., Carrington, M., Michaeli, S., Gull, K., and Rudenko, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN (Online):1932-6203
Published Online:26 October 2009
Copyright Holders:Copyright © 2009 Smith et al.
First Published:First published in PLoS ONE 4(10):e7532
Publisher Policy:Reproduced under a Creative Commons License

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