Regulation of a Novel Trypanosome RNA Binding Protein by Arginine Methylation

描述(申请人提供)：本项目的目标是确定精氨酸(Arg)甲基化调节动体寄生虫基因表达的机制。包括布氏锥虫、克鲁兹锥虫和利什曼原虫在内的动植体每年总共感染约2000万人，并导致数十万人死亡。目前严重缺乏安全有效的药物来对抗这些动质相关疾病，需要对寄生虫生物学有更深入的了解，以确定可以作为新的药物靶点的途径。动质体生物学的一个独特方面是明显缺乏对RNA聚合酶II的转录控制。在缺乏转录控制的情况下，基因调控主要依赖于转录后过程，如RNA的稳定性和翻译，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，限制性商业惯例是精氨酸甲基化的常见靶标，这种修饰可以显著影响目标限制性商业惯例的亚细胞定位和/或大分子相互作用。总之，这些观察结果表明，限制性商业惯例的精氨酸甲基化可能在动态体基因调控中发挥特别重要的作用。我们在布氏毛滴虫中鉴定了5种蛋白质精氨酸甲基转移酶(PRMTs)，鉴定了800多种精氨酸甲基转移酶。在本申请中，我们将研究一种新的Arg甲基化RBP，TbMiz1的功能。在体外，TbMiz1与RNA结合具有一定的序列特异性，但不与DNA结合。在体内，TbMiz1至少在三个Arg残基上发生甲基化，是布鲁氏毛滴虫生长所必需的。拟议的研究将开始评估TbMiz1的细胞功能，并阐明这些功能是如何受到Arg甲基化的影响的。TbMiz1含有一个C-末端富含Arg/甘氨酸(RG)的结构域，该结构域通常以Arg甲基化为靶点。已知的三种甲壳类仅限于这一地区。为了了解Arg甲基化的影响，我们将创建有条件地表达低甲基化TbMiz1突变体的布鲁氏锥虫细胞系，这些突变体要么缺乏整个RG结构域，要么在三个已知的甲基化部位含有赖氨酸到Arg的取代。在目标1中，我们将使用体外甲基化分析和体内相互免疫共沉淀(co-IP)来确定TbMiz1作为五种TbPRMT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来确定TbMiz1的亚细胞定位，并比较低甲基化突变体TbMiz1的定位。我们将通过串联亲和纯化的野生型TbMiz1的质谱学来鉴定TbMiz1的蛋白质结合伙伴，并通过质谱学和低甲基化突变体的共IP来确定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)来鉴定TbMiz1的体内RNA靶标。对低甲基化突变体进行RIP，然后对有效的目标RNA进行qRT-PCR，将揭示甲基化是否调节特定的TbMiz1-RNA相互作用。总体而言，拟议的实验将深入了解布氏毛滴虫中一个必要的RBP的功能，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.