Discussion

The mitochondrial ATP synthase is a bi-directional nanomotor, which in majority of eukaryotes works in its forward mode to synthesize ATP. The activity of this enzyme is negatively regulated by highly conserved inhibitory protein IF1. In Trypanosoma brucei, the mitochondrial ATP synthase switches its activity from the ATP production to ATP hydrolysis as the parasite differentiates from the insect procyclic stage (PF) to the mammalian bloodstream stage (BF). We identified T. brucei homolog of IF1 (TbIF1) and showed that on the protein level it is present only in PF cells and its expression is transiently upregulated during the differentiation from PF to metacyclic stage, the BF precursor. Upregulation of IF1 and ATP synthase inhibition is also observed in cancer cells, which undergo a metabolic switch from oxidative phosphorylation to glycolysis. Thus, TbIF1 can be involved in similar metabolic rewiring during the parasite’s differentiation. To comprehensively understand PF to BF metabolic transition, we study mechanisms underlying TbIF1 differential expression during the T. brucei life cycle. We showed that both steady state level and half-life of TbIF1 mRNA are dramatically increased in PF compared to BF, which in the context of absence of transcriptional regulation in T. brucei, indicates regulation by mRNA stability. Inhibition of translation results in TbIF1 mRNA accumulation in BF, which suggest existence of short-lived destabilizing RNA-binding protein. Using CAT-based reporters, we dissect TbIF1 UTRs to identify sequential and/or structural elements directing TbIF1 mRNA stability. The factors, which interact with the identified motifs, are being purified using Csy4, an inducible RNA hairpin-binding nuclease. Together, the data reveal mRNA-controlled mechanisms contributing to metabolic progression during the development of the infectious form of this parasite.