Discussion

Uniquely, amongst eukaryotes, Trypanosoma brucei RNA polymerase I (RNAPI) generates a subset of mRNAs. The most important of these gives rise to the highly abundant variant surface glycoprotein (VSG), the major surface protein of the pathogenic form of T. brucei, and key mediator of antigenic variation. Research on RNAPI transcriptional regulation in T. brucei has focused on understanding its role in monoallelic VSG transcription. However, relatively little attention has been paid to the regulation of ribosomal DNA (rDNA) array transcription. Our previous findings have suggested that only a subset of rDNA arrays are active and that changes in rDNA transcriptional activity may have an impact on VSG transcription. We aim to identify and characterize the trans-acting factors that regulate rDNA transcription in T. brucei, thereby ensuring optimal rRNA and VSG mRNA production. We have generated a set of reporter cell lines incorporating a neomycin phosphotransferase (NPT) gene either immediately downstream of an ectopic or native rDNA promoter (proximal) or adjacent to the 5.8S sequence within an rDNA array (promoter distal). Consistent with the previously identified expression variability seen between rDNA spacers, we observed variable reporter expression between arrays, whether the reporter was integrated in a promoter-proximal or -distal position. To enable the identification of factors responsible for rDNA array transcriptional repression, we introduced the RNAi library into a subset of our reporter lines and screened for enhanced resistance to G418, leading to the identification of several candidate factors. Efforts to validate these putative novel rDNA transcriptional regulators are currently underway.