Posttranslational modification of the regulatory RNA binding protein, ZFP3

调节性 RNA 结合蛋白 ZFP3 的翻译后修饰

• 批准号: 9298582
• 负责人: Laurie K. Read
• 金额: $ 19.94万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-17 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9298582
• 关键词: Aconitic Acid; Address; Affect; Arginine; Binding; Biochemical; Biological; Blood Circulation; Capsid Proteins; Cell Line; Cells; Cytoplasmic Granules; Data; Deposition; Development; Epitopes; Essential Genes; Gene Expression Regulation; Genetic Transcription; Genetic Translation; Goals; Immunofluorescence Immunologic; Immunoprecipitation; Insecta; Label; Laboratories; Length; Life Cycle Stages; Mass Spectrum Analysis; Measures; Medical; Messenger RNA; Methodology; Methylation; Modification; Molecular; Monitor; Morphology; Mutation; Nutrient; Organism; Parasites; Pathogenesis; Pattern; Peptides; Phosphorylation; Phosphoserine; Physiological; Polyribosomes; Population; Post-Translational Protein Processing; Post-Translational Regulation; Process; Proteins; Proteome; Proteomics; Quantitative Reverse Transcriptase PCR; RNA; RNA Polymerase II; RNA immunoprecipitation sequencing; RNA-Binding Proteins; Recruitment Activity; Regulation; Regulator Genes; Ribosomes; Role; Serine; Serine/Threonine Phosphorylation; Starvation; Stress; System; Testing; Threonine; Translations; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma procyclic acidic repetitive protein; Tyrosine; Variant; Yeasts; cohort; insight; mRNA Stability; messenger ribonucleoprotein; mutant; novel; overexpression; protein function; protein protein interaction; response; transcriptome sequencing; yeast two hybrid system

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.

RNA结合蛋白（RBP）与与基因调节相比对基因调节产生特别强大的影响。 其他生物体，因为动力质体不调节RNA聚合酶II转录，而是依靠 转录后基因调节机制。最近的蛋白质组学研究表明，许多t. brucei RBP受翻译后修饰（PTM）的约束，例如丝氨酸/苏氨酸磷酸化和 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； 但是，几乎一无所知，即PTM会影响动作塑料功能的机制 RBP。 ZFP3是一种调节性RBP，在哺乳动物血液形式（BF）T。Brucei和 刺激从BF到昆虫procyclic形式（PF）的分化。因此，ZFP3对于T. Brucei至关重要 生存和发病机理。多功能ZFP3结合并稳定数十个mRNA，刺激 通过PF特异性核糖体关联的EP1 procyclin mRNA的翻译，并募集到细胞质 饥饿应激期间，PF中的MRNP颗粒。我们实验室和其他人的蛋白质组学分析表明 14 kDa ZFP3包含两个甲基精氨酸和两个磷酸盐标记。在这里，我们建议测试 PTMS调节和多样化ZFP3功能的假设，从而导致其在BF和 PF T. Brucei。我们的初步数据表明精氨酸甲基化对于形态学至关重要 PF中ZFP3动作的表现称为“喷嘴”。在AIM 1中，我们将比较过表达发作的细胞 标记为野生型（WT）ZFP3，以表达那些过表达的低甲基化，降压型，甲基咪要或 磷酸化ZFP突变体。我们将测量ZFP3及其PTM变体对潜在BF的容量 PF分化，结合和调节特定mRNA的稳定性，刺激EP1 procyclin Translation和 调节ZFP3与核糖体，应力颗粒和其他结合伴侣的关联。我们也会表演 RNASEQ和RIPSEQ研究定义PTMS对ZFP3函数的全局影响。在AIM 2中，我们将量化 BF和PF中ZFP3上的PTM类，以全面了解该蛋白 在生命周期期间翻译后调节。使用新颖的，无标签的质谱法我们将 确定ZFP3含有甲基亚金氨酸磷serine/苏氨酸/酪氨酸，甲基lysine和 乙酸酯，我们将定义BF和PF寄生虫之间的差异。我们将检查能力 特定的PTM会影响彼此的沉积，导致具有不同PTM模式的ZFP分子 （“ PTM串扰”）采用一系列质谱方法，包括完整ZFP3的自上而下分析 分子。总的来说，拟议的研究将洞悉PTMS多样化的机制 并调节关键锥体调节RBP的功能。他们还将为 锥虫中的PTM串扰，并为其他关键的类似分析提供了方法学框架 锥虫RBP。