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

• 批准号: 323360091
• 负责人: Professorin Dr. Christine Elizabeth Clayton
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/323360091?language=en
• 关键词: Differentiation; Trypanosoma; brucei; master; regulator

African trypanosomes are parasitic unicellular flagellates. In a mammalian host, multiplying long slender bloodstream forms (at 37°C) convert to non-dividing short stumpy forms. After uptake into Tsetse, stumpy forms convert to dividing procyclic trypomastigotes, then epimastigotes, then mammalian-infective metacyclic forms. In culture, stumpy bloodstream forms convert to procyclic forms after addition of 3 mM cis-aconitate and a 27°C temperature shift.Remarkably, trypanosomes do not control transcription of individual protein-coding genes. Transcription is polycistronic and individual mRNAs are excised by processing. Gene expression is controlled by RNA-binding proteins that influence mRNA processing, translation, and decay. The RNA binding protein RBP10 is expressed in, and essential for survival of, long slender bloodstream forms, but is absent in stumpy forms and procyclics. Results from RNAi, induced expression, pull-down, RNA-Seq and tethering assays showed that RBP10 binds to mRNAs with the sequence UA(U)6 in their 3'-untranslated regions, causing translation repression and mRNA destruction. RBP10 targets include mRNAs encoding enzymes of procyclic energy metabolism, the major procyclic surface protein, 3 protein kinases, a protein phosphatase, and three procyclic-specific zinc finger RNA-binding proteins, ZC3H20, ZC3H21 and ZC3H22. Depletion of RBP10 in bloodstream forms, and transfer to 27°C, results in transformation to procyclic forms without cis-aconitate. Conversely, after induced or transient expression of RBP10 in procyclic forms and transfer to 37°C, some cells convert to bloodstream forms. (Without RBP10, they die.) together the results suggest that expression of RBP10 and a temperature of 37°C define bloodstream-form trypanosome identity.We here propose to investigate how expression of RBP10 itself is controlled, and the functions of downstream targets. We will first compare differentiation of established and freshly isolated trypanosome lines. New trypanosomes will be used as an experimental model if they differentiate more efficiently. To investigate how RBP10 expression is controlled we will define the RNA sequences required. To find candidate regulatory proteins, we will first identify all mRNA-binding proteins at different life-cycle stages, then combine the information with existing datasets, and use RNA affinity purification if feasible. Function will then be tested. We will investigate the roles of RBP10 phosphorylation and turnover in its function and regulation. To follow the differentiation cascade downstream of RBP10, we will analyse the functions of potential regulators whose expression is repressed by RBP10: the kinases and phosphatase, and the three zinc finger proteins.By the end of the project, the results, combined with those from complementary work, should yield a mechanistic understanding of how trypanosome developmental stages are maintained and how developmental switches are effected.

非洲锥虫是寄生的单细胞鞭毛虫。在哺乳动物宿主中，繁殖的细长血流形式（在37°C下）转化为不分裂的短短粗形式。在被采采蝇吸收后，矮胖的形式转化为分裂的前环锥鞭毛体，然后是上鞭毛体，然后是可感染的亚环形式。在培养中，在加入3 mM顺乌头酸盐和27°C的温度变化后，粗短的血流形式转化为前环形式。转录是多顺反子的，单个mRNA通过加工被切除。基因表达由RNA结合蛋白控制，RNA结合蛋白影响mRNA的加工、翻译和降解。RNA结合蛋白RBP 10在细长血流形式中表达，并且对于细长血流形式的存活是必需的，但是在短截形式和顺周期形式中不存在。来自RNAi、诱导表达、pull-down、RNA-Seq和tethering测定的结果表明，RBP 10与在其3 '非翻译区具有序列UA（U）6的mRNA结合，引起翻译抑制和mRNA破坏。RBP 10靶标包括编码前环能量代谢酶、主要前环表面蛋白、3种蛋白激酶、蛋白磷酸酶和3种前环特异性锌指RNA结合蛋白ZC 3 H20、ZC 3 H21和ZC 3 H22的mRNA。血流形式中的RBP 10耗尽并转移至27°C，导致转化为不含顺乌头酸的原环形式。相反，在诱导或瞬时表达原循环形式的RBP 10并转移至37°C后，一些细胞转化为血流形式。（如果没有RBP 10，他们就会死。总之，这些结果表明，RBP 10的表达和37°C的温度定义了血流形式的锥虫身份。我们在这里建议研究RBP 10本身的表达是如何控制的，以及下游靶点的功能。我们将首先比较建立的和新鲜分离的锥虫系的分化。如果新的锥虫分化更有效，它们将被用作实验模型。为了研究RBP 10表达是如何控制的，我们将定义所需的RNA序列。为了找到候选调控蛋白，我们将首先鉴定不同生命周期阶段的所有mRNA结合蛋白，然后将信息与现有数据集联合收割机结合，并在可行的情况下使用RNA亲和纯化。然后将测试功能。我们将研究RBP 10磷酸化和周转在其功能和调节中的作用。为了跟踪RBP 10下游的分化级联反应，我们将分析其表达被RBP 10抑制的潜在调节因子的功能：激酶和磷酸酶，以及三个锌指蛋白。在项目结束时，结合互补工作的结果，应该产生对锥虫发育阶段如何维持以及发育开关如何受到影响的机制性理解。