Function and regulation of the essential RNA binding protein, DRBD18

必需 RNA 结合蛋白 DRBD18 的功能和调节

基本信息

批准号：
10404501
负责人：
Laurie K. Read
金额：
$ 42.66万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-06 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10404501
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Address Affect Africa South of the Sahara African Trypanosomiasis Arginine Binding Binding Proteins Biochemical Biological Biological Assay Biological Process Biology Blood Circulation Cell physiology Data Development Disease Drug Targeting Event Gene Expression Gene Expression Regulation Genetic Genetic Translation Genomics Goals Homeostasis Human Immunoprecipitation Individual Insect Vectors Insecta Kinetoplastida Knowledge Laboratories Lead Livestock Mass Spectrum Analysis Mastigophora Mediating Medical Messenger RNA Methylation Modeling Molecular Morbidity - disease rate Nuclear Nuclear Export Nucleotides Oligonucleotides Open Reading Frames Organism Parasites Pathway interactions Pattern Peptide Initiation Factors Pharmaceutical Preparations Population Positioning Attribute Post-Transcriptional Regulation Post-Translational Protein Processing Process Protein Binding Domain Protein Kinase Proteins Proteomics RNA RNA-Binding Proteins Regulation Regulator Genes Regulatory Pathway Research Resolution Role Specificity Testing Transcript Translation Initiation Translations Trypanosoma Trypanosoma brucei brucei Tumor stage Vaccination Variant Virulence cohort combinatorial crosslink experimental study genome-wide human mortality in vivo insight mRNA Export mRNA Stability mRNA Transcript Degradation protein function protein protein interaction ribosome profiling transcriptome sequencing

项目摘要

ABSTRACT The flagellated protozoan, Trypanosoma brucei, is a devastating human and veterinary parasite in sub- Saharan Africa, and the causative agent of Human African Trypanosomiasis (HAT). HAT is fatal if untreated, vaccination is not an option, and available drugs are toxic, difficult to administer, and expensive. In the search for new treatments, understanding the basic biology of the parasite is a cornerstone on the path to discovery of unique biological processes that could potentially serve as drug targets. Trypanosomes are exceptional in that they perform gene regulation almost exclusively at posttranscriptional levels, through control of processes such as mRNA stability and translational efficiency. This reliance on posttranscriptional regulation necessitates that RNA binding proteins (RBPs) are the key effectors of trypanosome development, homeostasis, and virulence. Our laboratory discovered DRBD18, an abundant RBP that is essential for the survival of both the human bloodstream form (BF) and the insect vector procyclic form (PF) of T. brucei, and that is not conserved outside the Order Kinetoplastida. DRBD18 depletion in the PF results in significant changes in the abundance of nearly 1000 mRNAs, many of the most highly regulated themselves encoding RBPs and protein kinases. Thus, DRBD18 is positioned at the apex of numerous potential regulatory cascades. Proteomic data suggest that DRBD18 functions in both nuclear mRNA export and translation initiation. Remarkably, the ability of DRBD18 to stabilize or destabilize mRNAs as well as both its protein and mRNA binding specificities are dramatically regulated by arginine methylation, which acts as a molecular switch towards DRBD18 action. In the proposed studies, we will elucidate DRBD18 functions and regulation by 1) identifying direct DRBD18 mRNA targets, 2) mechanistically defining DRBD18 effector pathways, and 3) establishing the methylation-responsiveness of DRBD18 interactions and DRBD18 functions. In Aim 1, we will use iCLIP to define on a genome-wide level the sets of mRNAs that are directly bound by DRBD18 and to determine how these sets of bound mRNAs are regulated upon DRBD18 methylation. In Aim 2, we will determine the roles of DRBD18 in nuclear mRNA export and translation initiation, and test hypotheses regarding regulation of these functions by arginine methylation. In Aim 3, we will study combinatorial RBP interactions and test the hypothesis that methylation-sensitive interactions between DRBD18 and other RBPs regulate the specificity of DRBD18 mRNA targeting. Our studies will define distinct cis-trans modules that mediate methylation-sensitive DRDB18 functions. Using combined genetic, genomic, and biochemical approaches, the proposed studies will provide fundamental insights into specific gene regulatory events in T. brucei, and uncover regulatory mechanisms with wide- ranging applicability in trypanosomes. They also have the potential to broaden our understanding of RNA biology and its regulation by arginine methylation in higher organisms.

抽象的带鞭毛的原生动物布氏锥虫是一种对人类和兽医具有毁灭性的寄生虫。撒哈拉非洲，以及非洲人类锥虫病 (HAT) 的病原体。如果不治疗，HAT 是致命的，疫苗接种不是一种选择，而且可用的药物有毒、难以施用且昂贵。在搜索中对于新的治疗方法，了解寄生虫的基本生物学是发现寄生虫之路的基石独特的生物过程有可能作为药物靶点。锥虫的特殊之处在于它们几乎完全在转录后水平上进行基因调控，通过控制诸如如 mRNA 稳定性和翻译效率。这种对转录后调控的依赖使得 RNA 结合蛋白 (RBP) 是锥虫发育、稳态和毒力的关键效应器。我们的实验室发现了 DRBD18，这是一种丰富的 RBP，对于人类和人类的生存都至关重要。 T. brucei 的血流形式 (BF) 和昆虫媒介原环形式 (PF)，并且在外部不保守动质体目。 PF 中 DRBD18 的缺失导致近乎丰度的显着变化 1000 个 mRNA，其中许多受高度调控的自身编码 RBP 和蛋白激酶。因此， DRBD18 位于众多潜在监管级联的顶端。蛋白质组数据表明 DRBD18 在核 mRNA 输出和翻译起始中发挥作用。值得注意的是，DRBD18 的能力稳定或不稳定 mRNA 及其蛋白质和 mRNA 结合特异性都显着受精氨酸甲基化调节，精氨酸甲基化充当 DRBD18 作用的分子开关。在提议的研究中，我们将通过 1) 识别直接 DRBD18 mRNA 靶点，2) 来阐明 DRBD18 的功能和调控机械地定义 DRBD18 效应通路，以及 3) 建立 DRBD18 的甲基化响应性 DRBD18 相互作用和 DRBD18 功能。在目标 1 中，我们将使用 iCLIP 在全基因组水平上定义直接被 DRBD18 结合的 mRNA 组，并确定这些结合的 mRNA 组如何受 DRBD18 甲基化调节。在目标 2 中，我们将确定 DRBD18 在核 mRNA 输出中的作用和翻译起始，并测试有关精氨酸甲基化调节这些功能的假设。在目标 3 中，我们将研究组合 RBP 相互作用并检验甲基化敏感的假设 DRBD18 和其他 RBP 之间的相互作用调节 DRBD18 mRNA 靶向的特异性。我们的研究将定义介导甲基化敏感的 DRDB18 功能的不同顺反模块。使用结合遗传、基因组和生化方法，拟议的研究将提供基础深入了解 T. brucei 的特定基因调控事件，并揭示广泛的调控机制在锥虫中的广泛适用性。它们还有潜力拓宽我们对 RNA 的理解生物学及其在高等生物中精氨酸甲基化的调节。