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

本项目研究布氏锥虫锌指蛋白ZC 3 H11的功能。锥虫基因表达的大多数控制是转录后的。T.布氏杆菌在哺乳动物（血流形式）和采采蝇（原环形式）中生长。这两种形式都受到温度波动的影响：哺乳动物为36-40°C，采采蝇为20 - 40°C（理想情况下为27°C）。热休克抑制翻译，并导致降解，大多数mRNA，但有些，包括那些编码伴侣，是稳定的，并继续translated.In前期工作和最后一个阶段，我们表明，ZC 3 H11结合（AUU）重复在3 '-非翻译区的mRNA编码的伴侣复合物，重新折叠蛋白质后热休克。在血液形式中，ZC 3 H11是必不可少的;它通过一种复合物稳定结合的mRNA，该复合物包括四种其他必需蛋白：MKT 1，PBP 1，LSM 12和poly（A）结合蛋白。双杂交筛选和亲和纯化的结果表明，MKT 1是一个转录后调控网络的枢纽，包括与其他几种RNA结合蛋白和泛素化途径的相互作用。在前环形式中，ZC 3 H11在27°C下不需要，但在36°C以上的热休克反应和生存是必需的。相反。在任何温度下，MKT 1似乎都不需要在顺周期中。在正常温度下，两种形式的ZC 3 H11蛋白几乎检测不到，但在热休克时，蛋白质水平增加。热休克引起ZC 3 H11蛋白稳定性的适度增加，但调节主要是翻译：ZC 3 H11 mRNA主要存在于多核糖体中，仅在热休克后。ZC 3 H11在前循环热休克反应中是如何起作用的？这些结果对真核生物中mRNA翻译和降解的控制具有更广泛的意义。最初，我们将进行高通量筛选和蛋白质组学，以找到可能参与的其他蛋白质：RNAi筛选，以鉴定热休克反应所需的蛋白质和/或在27°C抑制ZC 3 H11 mRNA翻译;系链筛选，以鉴定可以转录后影响报告基因以前环形式表达的蛋白质;以及在各种条件下的串联亲和纯化，以重新评估ZC 3 H11的相互作用。为了分析ZC 3 H11 mRNA翻译的调节，我们将尽可能窄地定义ZC 3 H11 mRNA中的调节元件，并尝试以生物化学和/或使用筛选结果纯化相互作用的蛋白质。这些蛋白质的相互作用反过来又表明了翻译控制的机制。为了分析ZC 3 H11在前循环热休克反应中的作用机制，我们将首先使用进一步的遗传操作来确定MKT 1是否参与其中。如果是，我们将更详细地研究MKT 1的功能;如果不是，我们将从屏幕上调查替代候选人。

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.