RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMβ. After transfection at ∼5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced concanavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET-procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.
- Concanavalin A
- MALDI-TOF mass spectrometry
- RNA interference library
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)