Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters

Burkard, Gabriela Schumann; Jutzi, Pascal; Roditi, Isabel

doi:10.1016/j.molbiopara.2010.09.002

Cited by 217 publications

(197 citation statements)

References 14 publications

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“…Bloodstream-form T. brucei 2T1 cells were maintained and transfected as described [31] except that Tb-BSF buffer [54] was used for all transfections. The pRPaSce* plasmid was used to derive Sce* cells from 2T1 cells as described [31].…”

Section: Methodsmentioning

confidence: 99%

A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei

et al. 2014

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In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3′-untranslated region; they presumably interact with ribonucleases and translation factors. However, very few such proteins have been characterized in any detail. Here we describe a genome-wide screen to find proteins implicated in post-transcriptional regulation in Trypanosoma brucei. We made a library of random genomic fragments in a plasmid that was designed for expression of proteins fused to an RNA-binding domain, the lambda-N peptide. This was transfected into cells expressing mRNAs encoding a positive or negative selectable marker, and bearing the “boxB” lambda-N recognition element in the 3′-untranslated region. The screen identified about 300 proteins that could be implicated in post-transcriptional mRNA regulation. These included known regulators, degradative enzymes and translation factors, many canonical RNA-binding proteins, and proteins that act via multi-protein complexes. However there were also nearly 150 potential regulators with no previously annotated function, or functions unrelated to mRNA metabolism. Almost 50 novel regulators were shown to bind RNA using a targeted proteome array. The screen also provided fine structure mapping of the hit candidates' functional domains. Our findings not only confirm the key role that RNA-binding proteins play in the regulation of gene expression in trypanosomatids, but also suggest new roles for previously uncharacterized proteins.

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Section: Methodsmentioning

confidence: 99%

A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei

et al. 2014

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“…MEF cells were detached from the plate, and 2×10 6 cells were immediately resuspended in 100 µl of transfection buffer (50 mM HEPES [pH 7.3], 90 mM NaCl, 5 mM KCl, 0.15 mM CaCl 2 ) [35]. A maximal amount of 5 µg of plasmid DNA was added to the cell suspension, and electroporation was carried out using the Amaxa Nucleofector system (program # T-020).…”

Section: Methodsmentioning

confidence: 99%

Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

et al. 2014

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“…In procyclic forms, procyclin is required for robust infection and transmission through the tsetse fly (66). From a clinical perspective, parasite genes impacting the efficacy of all five currently available drugs have been identified and the majority of these encode surface-exposed proteins (67)(68)(69)(70)(71). Importantly, nearly all of these proteins were identified in the cell surface proteome, emphasizing the functional relevance of the surface proteome.…”

Section: Functional Implications Of the Cell Surface Proteomes Proteomentioning

confidence: 99%

Cell Surface Proteomics Provides Insight into Stage-Specific Remodeling of the Host-Parasite Interface in Trypanosoma brucei*

Shimogawa

Saada

Vashisht

et al. 2015

Molecular & Cellular Proteomics

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African trypanosomes are devastating human and animal pathogens transmitted by tsetse flies between mammalian hosts. The trypanosome surface forms a critical host interface that is essential for sensing and adapting to diverse host environments. However, trypanosome surface protein composition and diversity remain largely unknown. Here, we use surface labeling, affinity purification, and proteomic analyses to describe cell surface proteomes from insect-stage and mammalian bloodstreamstage Trypanosoma brucei. The cell surface proteomes contain most previously characterized surface proteins. We additionally identify a substantial number of novel proteins, whose functions are unknown, indicating the parasite surface proteome is larger and more diverse than generally appreciated. We also show stage-specific expression for individual paralogs within several protein families, suggesting that fine-tuned remodeling of the parasite surface allows adaptation to diverse host environments, while still fulfilling universally essential cellular needs. Our surface proteome analyses complement existing transcriptomic, proteomic, and in silico analyses by highlighting proteins that are surface-exposed and thereby provide a major step forward in defining the hostparasite interface. Molecular & Cellular

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Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters

Cited by 217 publications

References 14 publications

A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei

A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei

Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

Cell Surface Proteomics Provides Insight into Stage-Specific Remodeling of the Host-Parasite Interface in Trypanosoma brucei*

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