Posttranslational modification of the regulatory RNA binding protein, ZFP3

RNA结合蛋白（RBP）对动质体中的基因调控具有特别强的作用， 其他生物，因为动质体不调节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T。布氏 RBP经历翻译后修饰（PTM），如丝氨酸/苏氨酸磷酸化和丝氨酸/苏氨酸磷酸化。 精氨酸甲基化。在其他系统中，PTM扩展RBP功能并有助于其调节; 然而，关于PTM影响动质体功能的机制几乎一无所知， 限制性商业惯例。ZFP 3是哺乳动物血流形式（BF）T中必需的调节性RBP。布鲁氏菌和 刺激从BF分化为昆虫原环形式（PF）。因此，ZFP 3对T.布氏 生存和发病机制。多功能ZFP 3结合并稳定数十种mRNA， 通过PF特异性核糖体结合翻译EP 1前环素mRNA，并被募集到细胞质 饥饿应激期间PF中的mRNP颗粒。我们实验室和其他实验室的蛋白质组学分析表明， 14 kDa ZFP 3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PTM调节ZFP 3功能并使其多样化，从而有助于其在BF中的关键作用， PF T.布鲁塞。我们的初步数据表明，精氨酸甲基化是必不可少的形态 ZFP 3在PF中的作用表现为“喷嘴”。在目标1中，我们将比较过表达表位的细胞， 标记的野生型（WT）ZFP 3与过表达低甲基化、低磷酸化、甲基模拟物或 拟磷酸化ZFP突变体。我们将测量ZFP 3及其PTM变体增强BF的能力， PF分化，结合和调节特定mRNA的稳定性，刺激EP 1原环素翻译， 调节ZFP 3与核糖体、应激颗粒和其他结合伴侣的结合。我们还将表演 RNAseq和RIPseq研究，以确定PTM对ZFP 3功能的总体影响。目标2：量化 BF和PF中ZFP 3的PTM类别，以全面了解这种蛋白质的 生命周期中的翻译后调节。使用新的无标记质谱方法，我们将 确定携带甲基精氨酸磷酸丝氨酸/苏氨酸/酪氨酸、甲基赖氨酸的ZFP 3的分数，和 乙酰赖氨酸，我们将定义BF和PF寄生虫之间的差异。我们将研究 特定的PTM影响彼此的沉积，导致ZFP分子具有不同的PTM模式 （“PTM串扰”）使用一系列质谱方法，包括对完整ZFP 3进行自上而下的分析 分子。总的来说，拟议的研究将提供深入了解PTM多样化的机制 并调节锥虫关键调节RBP的功能。他们还将提供第一个洞察到 PTM串扰锥虫，并提供了一个方法框架，类似的分析，其他关键 锥虫RBP。

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.