Differentiation of Trypanosoma brucei: the master regulator RBP10 and its targets

• Applicant: Professorin Dr. Christine Elizabeth Clayton
• Subject Area: Cell Biology ; Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323360091

African trypanosomes are parasitic unicellular flagellates. In a mammalian host, multiplying long slender bloodstream forms (at 37°C) convert to non-dividing short stumpy forms. After uptake into Tsetse, stumpy forms convert to dividing procyclic trypomastigotes, then epimastigotes, then mammalian-infective metacyclic forms. In culture, stumpy bloodstream forms convert to procyclic forms after addition of 3 mM cis-aconitate and a 27°C temperature shift.Remarkably, trypanosomes do not control transcription of individual protein-coding genes. Transcription is polycistronic and individual mRNAs are excised by processing. Gene expression is controlled by RNA-binding proteins that influence mRNA processing, translation, and decay. The RNA binding protein RBP10 is expressed in, and essential for survival of, long slender bloodstream forms, but is absent in stumpy forms and procyclics. Results from RNAi, induced expression, pull-down, RNA-Seq and tethering assays showed that RBP10 binds to mRNAs with the sequence UA(U)6 in their 3'-untranslated regions, causing translation repression and mRNA destruction. RBP10 targets include mRNAs encoding enzymes of procyclic energy metabolism, the major procyclic surface protein, 3 protein kinases, a protein phosphatase, and three procyclic-specific zinc finger RNA-binding proteins, ZC3H20, ZC3H21 and ZC3H22. Depletion of RBP10 in bloodstream forms, and transfer to 27°C, results in transformation to procyclic forms without cis-aconitate. Conversely, after induced or transient expression of RBP10 in procyclic forms and transfer to 37°C, some cells convert to bloodstream forms. (Without RBP10, they die.) together the results suggest that expression of RBP10 and a temperature of 37°C define bloodstream-form trypanosome identity.We here propose to investigate how expression of RBP10 itself is controlled, and the functions of downstream targets. We will first compare differentiation of established and freshly isolated trypanosome lines. New trypanosomes will be used as an experimental model if they differentiate more efficiently. To investigate how RBP10 expression is controlled we will define the RNA sequences required. To find candidate regulatory proteins, we will first identify all mRNA-binding proteins at different life-cycle stages, then combine the information with existing datasets, and use RNA affinity purification if feasible. Function will then be tested. We will investigate the roles of RBP10 phosphorylation and turnover in its function and regulation. To follow the differentiation cascade downstream of RBP10, we will analyse the functions of potential regulators whose expression is repressed by RBP10: the kinases and phosphatase, and the three zinc finger proteins.By the end of the project, the results, combined with those from complementary work, should yield a mechanistic understanding of how trypanosome developmental stages are maintained and how developmental switches are effected.

• DFG Programme: Research Grants
• International Connection: Uganda
• International Co-Applicant: Professor Dr. Julius Mulindwa