Regulation of a Novel Trypanosome RNA Binding Protein by Arginine Methylation

描述（由申请人提供）：本项目的目的是确定精氨酸（arg）甲基化调节动质体寄生虫基因表达的机制。动质体包括布氏锥虫、T. cruzi，和利什曼原虫属，每年总共感染约2 000万人，并造成数十万人死亡。目前严重缺乏安全有效的药物来对抗这些动质体相关疾病，需要对寄生虫生物学有更深入的了解，以确定可能作为新药物靶点的途径。动质体生物学的一个独特方面是RNA聚合酶II的转录控制的明显缺乏。在缺乏转录调控的情况下，基因调控主要依赖于转录后过程，如RNA稳定性和翻译，从而使RNA结合蛋白（RBP）成为细胞功能的关键调节因子。在高等真核生物中，RBP是精氨酸甲基化的常见靶标，精氨酸甲基化是一种可以显著影响靶RBP的亚细胞定位和/或大分子相互作用的修饰。总之，这些观察结果表明，精氨酸甲基化的RBPs可以发挥特别重要的作用，在动质体基因调控。我们已经鉴定了五种蛋白质精氨酸甲基转移酶（PRMT），并在T.布鲁塞。在本申请中，我们将研究一种新的arg甲基化RBP，TbMiz 1的功能。在体外，TbMiz 1以一定的序列特异性结合RNA，但不结合DNA。在体内，TbMiz 1在至少三个arg残基上被甲基化，并且它对于原环形式T的生长是必需的。布鲁塞。拟议的研究将开始评估TbMiz 1细胞功能，并阐明这些功能如何受到精氨酸甲基化的影响。TbMiz 1含有一个C-末端精氨酸/富含甘氨酸（RG）结构域，这是一个通常针对精氨酸甲基化的结构域。三种已知的甲基大分子仅限于这一区域。为了了解arg甲基化的影响，我们将创建T。有条件地表达低甲基化TbMiz 1突变体的布氏细胞系，要么缺乏整个RG结构域，要么在三个已知的甲基区具有赖氨酸到Arg的取代。在目标1中，我们将使用体外甲基化测定和相互体内免疫共沉淀（co-IP）来确定TbMiz 1作为五种TbPRMT中的每一种的底物的能力。在目标2中，我们将通过免疫印迹和免疫荧光确定TbMiz 1亚细胞定位，并比较低甲基化突变体TbMiz 1的定位。我们将通过串联亲和纯化的野生型TbMiz 1的质谱鉴定TbMiz 1的蛋白结合伴侣，并通过低甲基化突变体的质谱和co-IP确定Arg甲基化是否影响这些相互作用。在目标3中，我们将使用RNA免疫沉淀和深度测序（RIP-seq）鉴定TbMiz 1的体内RNA靶标。低甲基化突变体的RIP，随后经验证的靶RNA的qRT-PCR将揭示甲基化是否调节特异性TbMiz 1-RNA相互作用。总的来说，拟议的实验将提供深入了解的功能，一个必要的RBP在T。布氏杆菌，并阐明精氨酸甲基化如何调节这些功能。

DESCRIPTION (provided by applicant): The goal of this project is to identify mechanisms by which arginine (arg) methylation regulates gene expression in kinetoplastid parasites. Kinetoplastids, including Trypanosoma brucei, T. cruzi, and Leishmania spp., collectively infect approximately 20 million people and cause hundreds of thousands of deaths per year. There is an appalling lack of safe and effective drugs to combat these kinetoplastid-related diseases, and a deeper understanding of parasite biology is required to identify pathways that could act as novel drug targets. One unique aspect of kinetoplastid biology is the apparent absence of transcriptional control of RNA polymerase II. In the absence of transcriptional control, gene regulation relies largely on posttranscriptional processes such as RNA stability and translation, thereby making RNA binding proteins (RBPs) critical regulators of cellular function. In higher eukaryotes, RBPs are common targets of arg methylation, a modification that can dramatically affect the subcellular localization and/or macromolecular interactions of target RBPs. Together, these observations suggest that arg methylation of RBPs could play an especially important role in kinetoplastid gene regulation. We have characterized five protein arg methyltransferases (PRMTs) and identified over 800 arg methylproteins in T. brucei. In this application, we will examine the function of a novel arg methylated RBP, TbMiz1. In vitro, TbMiz1 binds to RNA with some sequence specificity, but it does not bind DNA. In vivo, TbMiz1 is methylated on at least three arg residues, and it is essential for growth of procyclic form T. brucei. The proposed studies will begin to assess TbMiz1 cellular functions and elucidate how these functions are impacted by arg methylation. TbMiz1 contains a C-terminal arg/glycine-rich (RG) domain, a domain commonly targeted for arg methylation. The three known methylargs are confined to this region. To understand the affects of arg methylation, we will create T. brucei cell lines conditionally expressing hypomethylated TbMiz1 mutants either lacking the entire RG domain or harboring lysine to arg substitutions at the three known methylargs. In Aim 1, we will determine the ability of TbMiz1 to act as a substrate for each of the five TbPRMTs using both in vitro methylation assays and reciprocal in vivo co-immunoprecipitations (co-IPs). In Aim 2, we will determine TbMiz1 subcellular localization by immunoblotting and immunofluorescence and compare localization of the hypomethylated mutant TbMiz1. We will identify protein binding partners of TbMiz1 by mass spectrometry of tandem affinity purified wild type TbMiz1 and determine if arg methylation impacts these interactions by mass spectrometry and co-IP of the hypomethylated mutants. In Aim 3, we will identify in vivo RNA targets of TbMiz1 using RNA immunoprecipitation and deep sequencing (RIP-seq). RIP of the hypomethylated mutants followed by qRT-PCR of validated target RNAs will reveal whether methylation modulates specific TbMiz1-RNA interactions. Overall, the proposed experiments will provide insight into the functions of an essential RBP in T. brucei and elucidate how arg methylation can modulate these functions.