Function and regulation of the essential RNA binding protein, DRBD18

摘要 有鞭毛的原虫布氏锥虫是一种毁灭性的人类和兽医寄生虫，在亚热带地区 撒哈拉非洲，人类非洲锥虫病(HAT)的病原体。如果不治疗是致命的， 接种疫苗不是一种选择，而且现有的药物有毒、难以管理和昂贵。在搜索中 对于新的治疗方法，了解这种寄生虫的基本生物学是发现这种寄生虫的基石 可能成为药物靶标的独特生物过程。锥虫在这一点上是例外的 它们几乎完全在转录后水平上执行基因调控，通过控制 作为信使核糖核酸的稳定性和翻译效率。这种对转录后调控的依赖使得 RNA结合蛋白(RBPs)是锥虫发育、内环境稳定和毒力的关键效应因子。 我们的实验室发现了DRBD18，一种丰富的RBP，对人类和人类的生存都是必不可少的 布鲁氏毛滴虫的血流型(BF)和昆虫媒介原环型(PF)，且不是外界保守的 动植体目。DRBD18在PF中的枯竭导致近 1000个mRNAs，许多调节最高的自身编码限制性商业惯例和蛋白激酶。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输出和翻译启动中起作用。值得注意的是，DRBD18的能力 稳定或破坏mRNAs及其蛋白质和mna结合的特异性是显著的。 受精氨酸甲基化调节，这是向DRBD18作用的分子开关。在建议的 研究方面，我们将通过1)识别直接的DRBD18 mRNA靶标来阐明DRBD18的功能和调控，2) 机械地定义DRBD18效应通路，以及3)建立甲基化反应性 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 DRBD18直接结合的mRNA集，并确定这些结合的mRNA集是如何 受DRBD18甲基化调控。在目标2中，我们将确定DRBD18在核mRNA输出中的作用 和翻译启动，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RBP的组合相互作用，并检验甲基化敏感的假设 DRBD18与其他限制性商业惯例之间的相互作用调节着DRBD18 mRNA靶向的特异性。我们的 研究将定义不同的顺式-反式模块，这些模块介导甲基化敏感的DRDB18功能。vbl.使用 结合遗传、基因组和生化方法，拟议的研究将提供基本的 洞察布氏毛滴虫中特定的基因调控事件，并揭示具有广泛性的调控机制 在锥虫中的范围适用性。它们也有可能扩大我们对RNA的理解 高等生物体中的生物学及其精氨酸甲基化的调节。

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.