Kinetoplastid parasites (Leishmania, Trypanosoma brucei, and Trypanosoma cruzi) cause devastating diseases that afflict ~8.5 million people worldwide. These parasites experience multiple environmental stressors throughout their lifecycles, including substantial fluctuations in temperature, pH, and nutrient availability. The ability to sense and respond to these stresses, while essential for parasite survival, is poorly understood. Genetic tools for understanding important questions in Leishmania biology are currently quite limited, especially compared to those available for T. brucei. The goals of this proposal are 1) to develop and validate a novel genome-scale Gain-of-Function (GoF) overexpression library as a resource for the Leishmania research community; and 2) to use the GoF library to identify RNA binding proteins (RBPs) that regulate L. donovani and T. brucei purine transporters in response to purine starvation, which is a model nutrient sensing and stress response pathway. Purines are essential nutrients obtained exclusively from the host. Upon removal of purines from the growth medium, L. donovani promastigotes arrest in the G1 phase of the cell cycle and enter a quiescent-like state in which they can persist for over 90 days in culture. As part of this robust stress response, both L. donovani and T. brucei increase expression of cell surface purine transporters such as LdNT3 and TbNT8.1, respectively. The 3'-untranslated regions (UTRs) of the LdNT3 and TbNT8.1 mRNAs encode a conserved regulatory element, predicted to form stem-loop structures, that represses expression of the transporters when purines are abundant; this repression is relieved when purines are scarce. Surprisingly, the TbNT8.1 stem-loop does not function in Leishmania, even though the loops differ by only three nucleotides. This intimates that the orthologous RBPs that bind this conserved stem-loop have evolved different binding specificities. We predict that expressing the cognate T. brucei RBP in trans will confer repressor activity to the TbNT8.1 stem-loop in L. donovani . We propose to exploit this possibility to identify the TbNT8.1 stem-loop binding protein via functional complementation in Leishmania. To achieve this, in Aim 1 we will take advantage of the fact that a plasmid library encoding the majority of T. brucei open reading frames (the TbORFeome) was recently constructed. Using an efficient in vitro recombination reaction (Gateway), we will transfer the TbORFeome to vectors we designed for inducible expression in L. donovani (the Ld_TbORFeome library). This new library will be transfected into an L. donovani reporter cell line that will allow us to identify TbORFs in the library that bind to the TbNT8.1 stem-loop and repress expression of the reporter (Aim 2). Completion of these Specific Aims will not only identify an RBP that regulates part of a highly conserved and essential stress response, but will also provide the community with a novel and versatile...