Function and regulation of the essential RNA binding protein, DRBD18

摘要 有鞭毛的原生动物布氏锥虫是一种毁灭性的人类和兽医寄生虫， 撒哈拉非洲，以及非洲人类锥虫病（HAT）的病原体。HAT如果不治疗是致命的， 接种疫苗不是一种选择，现有的药物有毒，难以管理，而且昂贵。在搜索 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的基石。 这些独特的生物过程可能成为药物靶点。锥虫是个例外 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。这种对转录后调节的依赖需要， RNA结合蛋白（RBP）是锥虫发育、稳态和毒力的关键效应子。 我们的实验室发现了DRBD 18，这是一种丰富的RBP，对人类和哺乳动物的生存至关重要。 血流型（BF）和虫媒原环型（PF）。布氏杆菌，而这是不保守的外部 动质体目PF中的DRBD 18耗尽导致近100个基因的丰度发生显著变化。 1000个mRNA，其中许多高度调节的自身编码RBP和蛋白激酶。因此，在本发明中， DRBD 18位于许多潜在调控级联的顶点。蛋白质组学数据表明， DRBD 18在核mRNA输出和翻译起始中起作用。值得注意的是，DRBD 18 稳定或不稳定的mRNA以及它的蛋白质和mRNA结合特异性显着 由精氨酸甲基化调节，精氨酸甲基化充当DRBD 18作用的分子开关。拟议 研究中，我们将阐明DRBD 18的功能和调节1）确定直接DRBD 18 mRNA的目标，2） 机制上定义DRBD 18效应通路，以及3）建立DRBD 18的甲基化反应性。 DRBD 18相互作用和DRBD 18功能。在目标1中，我们将使用iCLIP在全基因组水平上定义 直接与DRBD 18结合的mRNA组，并确定这些结合的mRNA组如何与DRBD 18结合。 DRBD 18甲基化的调控。目的2：研究DRBD 18在细胞核mRNA输出中的作用 和翻译起始，并测试关于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，并测试甲基化敏感的假设， DRBD 18和其他RBP之间的相互作用调节DRBD 18 mRNA靶向的特异性。我们 研究将定义介导甲基化敏感性DRDB 18功能的不同顺-反模块。使用 结合遗传学，基因组学和生物化学方法，拟议的研究将提供基础 深入了解T.布氏杆菌，并揭示调控机制与广泛的， 范围适用于锥虫。它们也有可能拓宽我们对RNA的理解 生物学及其在高等生物中通过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.