Trypanosoma brucei expression-site-associated-gene-8 protein interacts with a Pumilio family protein

The expression site (ES) loci of Trypanosoma brucei are a valuable model for allelic exclusion and post-transcriptional regulation in a highly divergent eukaryote. ES exist to facilitate the expression and switching of the variant surface glycoproteins (VSG) that are central to trypanosome virulence and persistence. A collection of other potential virulence determinants, known as expression-site-associated-genes (ESAGs), are co-transcribed from the single upstream promoter. ESAGs may be involved in regulating the transcriptional state of the ES, as well as contributing additional surface proteins and receptors. We have previously shown that a putative regulatory protein, ESAG8, accumulates within the nucleolus, although 20% of the protein is cytoplasmic. Here we identify TbPUF1, a cytoplasmic ESAG8-interacting protein that falls into the Puf family of regulators of mRNA stability. Our experiments show that, as in other Puf family proteins, the most C-terminal repeats of TbPUF1 mediate its interaction with ESAG8. TbPUF1 is essential for cell viability, and preliminary results suggest that its overexpression seriously affects parasite virulence. T. brucei is the most evolutionary divergent organism in which a Puf family protein has been identified, and our initial experiments suggest that this protein may also regulate RNA stability in trypanosomes.