The regulation of mRNA processing in Trypanosoma brucei: kinetics, factors and mechanisms

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There is increasing evidence that all steps in the lifetime of eukaryotic mRNAs are interconnected: transcription, splicing, and polyadenylation mutually influence each other; nuclear events affect the fate of the mRNA in the cytoplasm. In this proposal we intend to study these phenomena in the protist Trypanosoma brucei. T. brucei offers the unique advantage that mRNA processing is independent of transcription, and the latter is constitutive: levels of mRNAs are regulated by the rates of processing and degradation alone. This drastically simplifies interpretation of results, and has enabled us to mathematically model the kinetics of mRNA creation and destruction at steady state. T. brucei is also a model system for Kinetoplastid parasites: these cause devastating diseases of humans and livestock, including Leishmaniasis in the Middle East. The two applicants have complementary expertise and have collaborated informally for many years. The Israeli group is a world leader in the field of Kinetoplastid trans splicing and the German group has a similar position for mRNA degradation. We now aim to combine forces to understand how trans-splicing and polyadenylation are coordinated and how both processes contribute to the steady-state level of mRNA and its stability. Results from both labs show that all three processes are tightly linked, and low-resolution results from the Israeli lab show that splicing factors can also influence polyadenylation and mRNA decay. The project has two aims: to quantitate the contributions of splicing and polyadenylation to the steady-state mRNA level, and to understand the roles of specific proteins in controlling and linking the two processes. We will combine unbiased high-throughput approaches with more conventional assays at the level of single proteins and mRNAs. The German group will contribute a novel forward genetic screen in trypanosomes, as well as yeast two-hybrid screening and kinetic expertise. The Israeli group will map the landscape of splicing factor RNA binding sites; high-throughput sequencing data for this, and for cell lines depleted of the factors, will allow us to quantitate their roles at the nucleotide level and about the contributions of each individual splicing factor to the transcriptome. Our project addresses a question in trypanosome biology that has been open for 2 decades, and has broad relevance to concerted regulation of nuclear and cytoplasmic events.