Regulation of a Novel Trypanosome RNA Binding Protein by Arginine Methylation

描述（由申请人提供）：本项目的目标是确定精氨酸（arg）甲基化调节着丝质体寄生虫基因表达的机制。着丝质体，包括布氏锥虫、克氏锥虫和利什曼原虫，每年总共感染大约2000万人，造成数十万人死亡。目前缺乏安全有效的药物来对抗这些与着丝质体相关的疾病，需要对寄生虫生物学有更深入的了解，以确定可能作为新型药物靶点的途径。着丝质体生物学的一个独特方面是明显缺乏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arg甲基化的常见靶标，这种修饰可以显著影响靶rbp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rbp的arg甲基化可能在着丝质体基因调控中发挥特别重要的作用。我们鉴定了5个蛋白质精氨酸甲基转移酶（PRMTs），并鉴定了800多个精氨酸甲基化蛋白。在这个应用中，我们将研究一种新的arg甲基化RBP TbMiz1的功能。在体外，TbMiz1以一定的序列特异性与RNA结合，但不与DNA结合。在体内，TbMiz1至少有三个残基甲基化，这对原环型布鲁氏菌的生长至关重要。拟议的研究将开始评估TbMiz1细胞功能，并阐明这些功能如何受到arg甲基化的影响。TbMiz1含有一个c端富含精氨酸/甘氨酸（RG）结构域，这是一个通常用于精氨酸甲基化的结构域。已知的三种甲基化合物都局限在这个区域。为了了解精氨酸甲基化的影响，我们将创建有条件地表达低甲基化的TbMiz1突变体的布鲁氏T.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位点上含有赖氨酸来取代精氨酸。在目的1中，我们将使用体外甲基化测定和体内互反共免疫沉淀（co-IPs）来确定TbMiz1作为五种TbPRMTs的底物的能力。在Aim 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野生型TbMiz1的质谱鉴定TbMiz1的蛋白结合伙伴，并通过质谱和低甲基化突变体的共ip确定arg甲基化是否影响这些相互作用。在Aim 3中，我们将使用RNA免疫沉淀和深度测序（RIP-seq）鉴定TbMiz1的体内RNA靶点。对低甲基化突变体进行RIP，然后对验证的靶rna进行qRT-PCR，将揭示甲基化是否调节特异性的TbMiz1-RNA相互作用。总的来说，这些实验将提供对布鲁氏菌中基本RBP功能的深入了解，并阐明arg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.