RNA interference (RNAi) is an evolutionarily conserved pathway that contributes to the maintenance of eukaryotic genome integrity. One of the most ancient organisms in which RNAi has been described is the African trypanosome, Trypanosoma brucei, a protozoan parasite and the etiologic agent of African sleeping sickness. Previous results had shown that RNAi-deficiency in T. brucei leads to an upregulation of retrotransposon transcripts, in particular the Spliced Leader (SL)-associated non-LTR retrotransposon SLACS. I took advantage of these RNAi-deficient cells to investigate the mechanism of SLACS expression and to further understand the role of the RNAi machinery in regulating SLACS expression. My results indicate that RNAi-deficiency leads to rearrangements of SLACS-containing genomic loci, suggesting that the RNAi pathway, specifically the Argonaute protein Ago1, is important for preserving the stability of the T. brucei genome. Two populations of SLACS transcripts persist in these RNAi-deficient cells; a population of full-length transcripts and a heterogenous pool of smaller transcripts, which originate from SLACS ORF1, are associated with proteins, and may be double-stranded in vivo. Furthermore, SLACS transcription initiates from the +1 nucleotide of the interrupted SL RNA gene and is dependent on the activity of an upstream SL promoter.

I also characterized a role for a novel Dicer-like protein (Dcl2) in the T. brucei RNAi pathway. Dcl2 is a nuclear protein that can cleave a double-stranded RNA substrate in vitro. Much in the same way that Ago1 ablation induces RNAi-deficiency, knocking out Dcl2 leads to an upregulation of retrotransposon and repeat-derived transcripts, providing evidence that Dcl2 contributes to RNAi-dependent silencing in these organisms. While both Dcl2 and the cytoplasmic Dicer protein Del1 generate retrotransposon-derived small interfering RNAs (siRNAs), Dcl2 specifically generates repeat-derived siRNAs, pointing to a role for Dcl2 in the T. brucei nuclear RNAi pathway. Taken together, my results indicate that T. brucei, by expressing two Dicer enzymes with distinct subcellular localizations, has uniquely evolved a means of compartmentalizing the RNAi pathway between its nucleus and cytoplasm.