The synthesis and role of Trypanosoma brucei ZC3H11 during the heat shock response
布氏锥虫ZC3H11的合成及其在热激反应中的作用
基本信息
- 批准号:218732757
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:德国
- 项目类别:Research Grants
- 财政年份:2012
- 资助国家:德国
- 起止时间:2011-12-31 至 2018-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
本课题研究布鲁氏锥虫锌指蛋白ZC3H11的功能。锥虫基因表达的大部分控制是转录后的。布氏体在哺乳动物(血流型)和采采蝇(前循环型)中生长。这两种形式都受温度波动的影响:哺乳动物为36-40°C,采采蝇为20 -40°C(理想情况下为27°C)。热休克抑制大多数mrna的翻译,并导致其降解,但一些mrna,包括编码伴侣蛋白的mrna,是稳定的,并继续被翻译。在前期工作和最后一段时间,我们发现ZC3H11结合在mrna的3'-非翻译区(AUU)重复序列上,这些重复序列编码在热休克后重新折叠蛋白质的伴侣复合物。在血液形态中,ZC3H11是必不可少的;它通过一个复合物来稳定结合的mrna,该复合物包括其他四种必需蛋白:MKT1、PBP1、LSM12和poly(a)结合蛋白。2杂交筛选和亲和纯化的结果表明,MKT1处于转录后调控网络的中心,包括与其他几种rna结合蛋白和泛素化途径的相互作用。在顺环形式中,ZC3H11在27°C时不需要,但对于热休克反应和36°C以上的生存是必需的。相比之下。在任何温度下,似乎都不需要MKT1。在正常温度下,两种形式的ZC3H11蛋白几乎检测不到,但在热休克时,蛋白质水平增加。热休克导致ZC3H11蛋白稳定性适度升高,但主要是通过翻译调控:ZC3H11 mRNA主要存在于热休克后的多聚体中。我们的目标是回答两个主要问题:ZC3H11的表达是如何以顺环形式调节的?ZC3H11在顺循环热休克反应中是如何作用的?该结果对真核生物mRNA翻译和衰变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,以发现可能涉及的其他蛋白质:RNAi筛选,以确定在27°C下热休克反应和/或抑制ZC3H11 mRNA翻译所需的蛋白质;一种用于鉴定转录后影响报告基因表达的蛋白的栓系筛选法;和串联亲和纯化在不同条件下重新评估ZC3H11的相互作用。为了分析ZC3H11 mRNA翻译的调控,我们将尽可能狭窄地定义ZC3H11 mRNA中的调控元件,并尝试生化纯化相互作用蛋白,或使用筛选结果。这些蛋白的相互作用应该反过来表明翻译控制的机制。为了分析ZC3H11在顺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MKT1是否参与其中。如果是,我们将更详细地研究MKT1的功能;如果不是,我们将从筛选中研究替代候选。
项目成果
期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Professorin Dr. Christine Elizabeth Clayton其他文献
Professorin Dr. Christine Elizabeth Clayton的其他文献
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{{ truncateString('Professorin Dr. Christine Elizabeth Clayton', 18)}}的其他基金
Diversity in cap-binding translation factor complexes: roles in translation initiation and mechanisms of mRNA selection
帽结合翻译因子复合物的多样性:翻译起始中的作用和 mRNA 选择机制
- 批准号:
419208155 - 财政年份:2019
- 资助金额:
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Differentiation of Trypanosoma brucei: the master regulator RBP10 and its targets
布氏锥虫的分化:主调节因子 RBP10 及其靶标
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323360091 - 财政年份:2016
- 资助金额:
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Research Grants
The regulation of mRNA processing in Trypanosoma brucei: kinetics, factors and mechanisms
布氏锥虫 mRNA 加工的调控:动力学、因素和机制
- 批准号:
290543715 - 财政年份:2016
- 资助金额:
-- - 项目类别:
Research Grants
Post-transcriptional repression of gene expression in trypanosomes: roles of RNA-binding proteins in translation and mRNA decay
锥虫基因表达的转录后抑制:RNA 结合蛋白在翻译和 mRNA 衰减中的作用
- 批准号:
268445533 - 财政年份:2015
- 资助金额:
-- - 项目类别:
Research Grants
Complexity and function of messenger ribonucleoprotein particles
信使核糖核蛋白颗粒的复杂性和功能
- 批准号:
197040498 - 财政年份:2011
- 资助金额:
-- - 项目类别:
Research Grants
The transcriptome of human sleeping sickness
人类昏睡病的转录组
- 批准号:
163515193 - 财政年份:2010
- 资助金额:
-- - 项目类别:
Research Grants
The role of Pumilio-domain proteins in Trypanosoma brucei
Pumilio 结构域蛋白在布氏锥虫中的作用
- 批准号:
152065058 - 财政年份:2009
- 资助金额:
-- - 项目类别:
Research Grants
Globalanalyse von cis-Elementen und trans-Faktoren für die Regulation der mRNA-Abundanz während der Stadiendifferenzierung von Trypanonsoma brucei
布氏锥虫分化阶段mRNA丰度调节的顺式元件和反式元件的整体分析
- 批准号:
5396237 - 财政年份:2003
- 资助金额:
-- - 项目类别:
Research Grants
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