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

越来越多的证据表明，真核生物mRNA生命周期中的所有步骤都是相互关联的：转录、剪接和多聚腺苷酸化相互影响;核事件影响mRNA在细胞质中的命运。在这个建议中，我们打算研究这些现象中的原生生物布氏锥虫。T.布氏杆菌提供了独特的优势，即mRNA加工不依赖于转录，而后者是组成型的：mRNA的水平仅由加工和降解的速率调节。这大大简化了结果的解释，并使我们能够在稳态下对mRNA的产生和破坏的动力学进行数学建模。T.布鲁氏菌也是动质体寄生虫的模型系统：这些寄生虫引起人类和牲畜的毁灭性疾病，包括中东的利什曼病。这两个申请人具有互补的专门知识，多年来一直进行非正式合作。以色列小组是动质体反式剪接领域的世界领导者，德国小组在mRNA降解方面也有类似的立场。我们现在的目标是联合收割机的力量，以了解如何反式剪接和多聚腺苷酸化是协调的，以及这两个过程如何有助于mRNA的稳态水平及其稳定性。来自两个实验室的结果表明，所有三个过程是紧密相连的，来自以色列实验室的低分辨率结果表明，剪接因子也可以影响多聚腺苷酸化和mRNA衰变。该项目有两个目标：定量剪接和多聚腺苷酸化对稳态mRNA水平的贡献，并了解特定蛋白质在控制和连接这两个过程中的作用。我们将联合收割机无偏高通量方法与更常规的单蛋白和mRNA水平的检测方法相结合。德国小组将贡献一种新的锥虫正向遗传筛选，以及酵母双杂交筛选和动力学专业知识。以色列小组将绘制剪接因子RNA结合位点的地图;高通量测序数据，以及缺乏这些因子的细胞系，将使我们能够在核苷酸水平上定量它们的作用，以及每个剪接因子对转录组的贡献。我们的项目解决了锥虫生物学中的一个问题，这个问题已经开放了20年，并且与核和细胞质事件的协调调节具有广泛的相关性。

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.