Posttranslational modification of the regulatory RNA binding protein, ZFP3

RNAbindingProteins(RBPs)对动质体内的基因调控起着特别强的作用 其他生物，因为动质体并不调节RNA聚合酶II的转录，而是依赖于 转录后基因调控机制。最近的蛋白质组学研究表明，许多布氏毛滴虫 限制性商业惯例受到翻译后修饰(PTM)的影响，如丝氨酸/苏氨酸磷酸化和 精氨酸甲基化。在其他系统中，PTMS扩展了RBP功能，并有助于其调节； 然而，关于PTMS影响动态体功能的机制几乎一无所知 限制性商业惯例。ZFP3是一种调节性RBP，在哺乳动物血流形式(BF)、布氏支原体和 刺激BF向昆虫原环型(PF)的分化。因此，ZFP3对布氏毛滴虫至关重要。 生存和发病机制。多功能ZFP3结合并稳定数十个mRNAs，刺激 通过Pf特异性核糖体结合翻译EP1原环素mRNA，并被招募到细胞质中 饥饿应激过程中PF中MRNP颗粒的表达。来自我们实验室和其他实验室的蛋白质组分析表明 14 kDa ZFP3含有两个甲基精氨酸和两个磷酸丝氨酸标记。在这里，我们建议测试 假设PTMS调节和多样化ZFP3功能，从而有助于其在BF和BF中的关键作用 Pf T.brucei.我们的初步数据表明，精氨酸甲基化对于形态上的 ZFP3作用在PF中的表现称为“喷嘴”。在目标1中，我们将比较过度表达表位的细胞 标记野生型(WT)ZFP3到那些过度表达低甲基化、低磷化、甲基模拟或 磷酸化ZFP突变体。我们将测量ZFP3及其Ptm变体的能力，以增强BF PF分化，结合和调节特定mRNAs的稳定性，刺激EP1原环素翻译，以及 调节ZFP3与核糖体、应激颗粒和其他结合伙伴的联系。我们还将表演 RNAseq和RIPseq进行研究，以确定PTMS对ZFP3功能的全球影响。在目标2中，我们将量化 BF和PF中ZFP3上的PTM分类有助于对该蛋白的全面理解 生命周期中的翻译后调节。使用新的、无标记的质谱学方法，我们将 测定含有甲基精氨酸磷酸丝氨酸/苏氨酸/酪氨酸、甲基赖氨酸和 乙酰赖氨酸，我们将定义BF和PF寄生虫之间的区别。我们将研究其能力 特定的PTM相互影响彼此的沉积，导致ZFP分子具有不同的PTM模式 (“PTM串扰”)使用一系列的质谱学方法，包括自上而下分析完整的ZFP 3 分子。总的来说，拟议的研究将使人们深入了解临时技术转让多样化的机制。 并调节关键的锥虫调节RBP的功能。他们还将提供对以下方面的初步见解 锥体中的PTM串扰，并为类似的分析其他关键的 锥虫限制性商业惯例。

RNA binding proteins (RBPs) exert an especially strong effect on gene regulation in kinetoplastids compared to other organisms since kinetoplastids do not regulate RNA polymerase II transcription and instead rely on posttranscriptional gene regulatory mechanisms. Recent proteomic studies revealed that many T. brucei RBPs are subject to posttranslational modifications (PTMs) such as serine/threonine phosphorylation and arginine methylation. In other systems, PTMs expand RBP function and contribute to their regulation; however, almost nothing is known about the mechanisms by which PTMs impact the functions of kinetoplastid RBPs. ZFP3 is a regulatory RBP that is essential in mammalian bloodstream form (BF) T. brucei and stimulates differentiation from the BF to the insect procyclic form (PF). ZFP3 is, thus, critical for T. brucei survival and pathogenesis. The multifunctional ZFP3 binds and stabilizes dozens of mRNAs, stimulates translation of EP1 procyclin mRNA through PF-specific ribosome association, and is recruited to cytoplasmic mRNP granules in PF during starvation stress. Proteomic analyses from our lab and others showed that the 14 kDa ZFP3 contains two methylarginine and two phosphoserine marks. Here, we propose to test the hypothesis that PTMs regulate and diversify ZFP3 functions, thereby contributing to its critical roles in BF and PF T. brucei. Our preliminary data indicate that arginine methylation is essential for the morphological manifestation of ZFP3 action in PF termed “nozzle”. In Aim 1, we will compare cells that overexpress epitope tagged wild type (WT) ZFP3 to those overexpressing hypomethylated, hypophosphorylated, methylmimic, or phosphomimic ZFP mutants. We will measure the capacity of ZFP3 and its PTM variants to potentiate BF to PF differentiation, bind and modulate the stabilities of specific mRNAs, stimulate EP1 procyclin translation, and regulate ZFP3 association with ribosomes, stress granules, and other binding partners. We will also perform RNAseq and RIPseq studies to define the global impacts of PTMs on ZFP3 function. In Aim 2, we will quantify classes of PTMs on ZFP3 in BF and PF towards a comprehensive understanding of this protein's posttranslational regulation during the life cycle. Using novel, label-free mass spectrometry approaches we will determine the fraction of ZFP3 harboring methylarginine phosphoserine/threonine/tyrosine, methyllysine, and acetylysine, and we will define differences between BF and PF parasites. We will examine the capacity of specific PTMs to affect each others' deposition, leading to ZFP molecules harboring distinct PTM patterns (“PTM crosstalk”) using a range of mass spectrometry approaches, including top-down analysis of intact ZFP3 molecules. Collectively, the proposed studies will provide insight into the mechanisms by which PTMs diversify and modulate the functions of a key trypanosome regulatory RBP. They will also provide the first insights into PTM crosstalk in trypanosomes and provide a methodological framework for similar analyses of other critical trypanosome RBPs.