Project name: Trypanosoma brucei

Description: In trypanosomes, the apparent lack of regulation of RNA polymerase II-dependent transcription initiation poses a challenge to understand how these eukaryotes adjust gene expression in order to adapt to the contrasting environments they find during their life cycles. Evidence so far indicates that mRNA turnover and translation are the major control points in which regulation is exerted in trypanosomes. However, very little is known about which proteins are involved, and how do they regulate the abundance and translation of different mRNAs in different life stages. In this work, an RNA-binding protein, TbDRBD3, has been identified by affinity chromatography, and its function addressed using RNA interference, microarray analysis and immunoprecipitation of mRNA-protein complexes. The results obtained indicate that TbDRBD3 binds to a subset of developmentally regulated mRNAs encoding membrane proteins and intermediate metabolism enzymes, and that this association promotes the stabilization of the target transcripts. These observations raise the possibility that TbDRBD3-mRNA complexes act as a post-transcriptional operon, and provide a framework to interpret how trypanosomes regulate gene expression in the absence of transcriptional control.Keywords: Effect of depletion of an RNA-binding protein on the transcriptomeOverall design: In order to analyze whether TbDRBD3 has a role in the regulation of mRNA turnover, the transcriptome of procyclic trypanosomes depleted of TbDRBD3 was compared to that of uninduced cells using hybridizations of genomic microarrays. RNA samples were isolated from cells harvested after 48 hours of induction with tetracycline. At this point, cell growth was barely affected and trypanosomes remained highly motile. 15 µg of RNA were reverse-transcribed in the presence of 100 ng of oligo(dT)12-18, 0.25 mM dATP, 0.25 mM dTTP, 0.25 mM dGTP, 0.05 mM dCTP, 0.05 mM Cy3- or Cy5-dCTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 40 U of RNaseOUT (Invitrogen) and 400 U of Superscript III reverse transcriptase (Invitrogen). Reactions were incubated for 3 hours at 50ºC, stopped by heating at 70ºC for 15 min and treated with 5 U of RNase H for 20 min at 37ºC. cDNA was purified using the MinElute kit (Qiagen), ethanol precipitated and resuspended in hybridization buffer. Genomic T. brucei microarray glass slides containing ca. 24,000 independent random genomic clones (Brems et al., 2005) were pre-hybridized and hybridized as described (Diehl et al., 2002). Four hybridizations (two biological replicates with a dye swap each) were analyzed.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Release date: 2008-07-15

Last updated: 2007-08-09