The synthesis and role of Trypanosoma brucei ZC3H11 during the heat shock response

• Applicant: Professorin Dr. Christine Elizabeth Clayton
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Cell Biology
• Term: from 2012 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 218732757

This project concerns the function of the Trypanosoma brucei zinc-finger protein ZC3H11. Most control of trypanosome gene expression is post-transcriptional. T. brucei grows in mammals (bloodstream form) and in Tsetse flies (procyclic form). Both forms are subject to temperature fluctuations: 36-40°C in mammals and 20°C - 40°C (ideally, 27°C) in Tsetse flies. Heat shock inhibits translation of, and causes degradation of, most mRNAs, but some, including those encoding chaperones, are stable and continue to be translated.In preliminary work and the last period we showed that ZC3H11 binds to (AUU) repeats in the 3'-untranslated regions of mRNAs that encode the chaperone complexes that re-fold proteins after heat shock. In bloodstream forms, ZC3H11 is essential; it stabilizes bound mRNAs via a complex that includes four other essential proteins: MKT1, PBP1, LSM12 and poly(A) binding protein. Results from 2-hybrid screening and affinity purification suggest that MKT1 is at the hub of a post-transcriptional regulatory network, including interactions with several other RNA-binding proteins and the ubiquitination pathway.In procyclic forms, ZC3H11 is not required at 27°C, but is necessary for the heat shock response and for survival above 36°C. In contrast. MKT1 does not seem to be needed in procyclics at any temperature. At normal temperatures for both forms, ZC3H11 protein is barely detectable, but upon heat shock the protein level increases. Heat shock causes a modest increase in ZC3H11 protein stability, but the regulation is mainly of translation: ZC3H11 mRNA is mainly found in the polysomes only after heat shock.We aim to answer two main questions: How is expression of ZC3H11 regulated in procyclic forms? And how does ZC3H11 act during the procyclic heat shock response? The results have wider implications for control of mRNA translation and decay in eukaryotes.Initially we will conduct high-throughput screens and proteomics to find additional proteins that might be involved: an RNAi screen in order to identify proteins that are required for the heat shock response and/or suppress ZC3H11 mRNA translation at 27°C; a tethering screen to identify proteins that can post-transcriptionally influence reporter expression in procyclic forms; and tandem affinity purifications under various conditions to reassess interactions of ZC3H11. To analyse regulation of ZC3H11 mRNA translation, we will define the regulatory element in ZC3H11 mRNA as narrowly as possible, and attempt to purify interacting proteins biochemically, and/or using the screening results. Interactions of these proteins should in turn indicate the mechanism of translation control. To analyse the mechanism of action of ZC3H11 in the procyclic heat shock response, we will first use further genetic manipulation to determine whether MKT1 is involved. If it is, we will study the function of MKT1 in more detail: if not, we will investigate alternative candidates from the screens.

• DFG Programme: Research Grants