Functions of nuclear non-canonical poly(A) polymerases in Trypanosomes

描述（由申请人提供）：锥虫是一种血鞭毛寄生原生动物，在进化早期从其他真核生物中分化出来，在基因表达、能量代谢和其他细胞过程的调节方面具有许多独特的特征。确保哺乳动物和昆虫宿主适应环境条件的基因表达模式不包括蛋白质编码基因上RNA聚合酶II （Pol II）活性的转录起始控制。可能发生在两条DNA链上的混杂转录对细胞，特别是对具有活性RNA干扰机制的物种，应该是有毒的。为了解决这一矛盾，我们提出了一个假设，即在锥虫体内存在一个核多聚腺苷化导向/外泌体介导的RNA监测系统。我们的目标是结合转录组和蛋白质相互作用网络分析，以确定不同的非典型多聚(A)聚合酶1 （ncPAP1）复合物是否标记了核外泌体降解32-52的特定RNA类别。我们建议：1)。在体内鉴定ncPAP1的RNA靶点。布氏体基因组编码两种典型的核聚(A)聚合酶，可能催化mRNA聚腺苷化。在初步的研究中，我们已经鉴定出了ncPAP1和ncPAP2，这是一种对寄生虫来说不是必需的进展性PAP。工作模型是，反义转录物，可能还有其他转录物，被ncPAP1腺苷化，从而成为降解的目标。由于我们无法预测ncap1的具体靶点，因此将采用两种全球方法来鉴定体内与ncap1结合的rna并评估转录组的变化。2）. 定义ncPAP1蛋白相互作用网络。单个ncPAP1多肽酶活性不高，很可能需要RNA结合亚基来发挥其PAP活性。我们在亲和纯化的ncPAP1复合体中鉴定了四种RNA结合蛋白、Mtr4解旋酶和第二种假定的DEAD盒解旋酶，但没有外显体成分。我们打算评估以下假设：1)这些RNA结合蛋白中的每一个都与ncPAP1形成特定的复合物；2) ncPAP1聚腺苷化转录本通过RNA解旋酶靶向外泌体。公共卫生相关性：锥虫病是世界各地发展中国家主要寄生虫病的病原体。考虑到驻扎在波斯湾和阿富汗的美军中内脏利什曼病的发生，对有效的抗锥虫治疗的需求可能会在美国出现。现有的治疗方法往往是有毒和无效的，这就产生了发现新药的需求。针对必需的寄生虫特异性酶，如本研究中介绍的核非规范聚(A)聚合酶，是创造新一代锥虫的一种有前途的方法。

DESCRIPTION (provided by applicant): Trypanosomes are hemoflagellate parasitic protozoans that diverged early in evolution from other eukaryotes and possess many unique features with respect to regulation of gene expression, energy metabolism, and other cellular processes. The gene expression patterns that ensure adaptation to environmental conditions in mammalian and insect hosts do not include transcription initiation control of RNA polymerase II (Pol II) activity on protein-encoding genes. The promiscuous transcription, which presumably occurs on both DNA strands, should be toxic to cells, especially for species with active RNA interference machinery. To resolve this paradox, we put forward a hypothesis that a nuclear polyadenylation-directed/exosome-mediated RNA surveillance system exists in Trypanosomatids. Our goal is to combine transcriptome and protein interaction network analyses to determine whether the distinct non-canonical poly(A) polymerase 1 (ncPAP1) complexes mark specific RNA classes for 32-52 degradation by the nuclear exosome. We propose to: 1). Identify RNA targets of ncPAP1 in vivo. The genome of T. brucei encodes two canonical nuclear poly(A) polymerases, which presumably catalyze mRNA polyadenylation. In preliminary studies, we have identified ncPAP1, the main subject of this application, and related ncPAP2, which is a processive PAP that is nonessential for parasites. The working model is that antisense, and probably other transcripts, are adenylated by ncPAP1 and thus targeted for degradation. Because we cannot predict specific targets of ncPAP1, two global approaches will be undertaken to identify RNAs bound to ncPAP1 in vivo and assess changes in the transcriptome. 2). Define protein interaction network for ncPAP1. The individual ncPAP1 polypeptide is enzymatically inactive and most likely requires an RNA binding subunit to exert its PAP activity. We have identified four RNA binding proteins, Mtr4 helicase, and a second putative DEAD box helicase in affinity-purified ncPAP1 complex, but no exosome components. We intend to assess hypotheses that 1) each of these RNA binding proteins forms a specific complex with ncPAP1; and 2) transcripts polyadenylated by ncPAP1 are targeted to the exosome via RNA helicases. PUBLIC HEALTH RELEVANCE: Trypanosomatids are the causative agents of major parasitic diseases in developing countries around the world. Considering the occurrence of visceral leishmaniasis among U.S. troops stationed in the Persian Gulf and Afghanistan, a demand for effective anti-trypanosomal therapies may be anticipated in the United States. Available treatments are often toxic and ineffective, creating demand for the discovery of new drugs. Targeting essential parasite-specific enzymes, such as nuclear non-canonical poly(A) polymerase introduced in our application, is a promising approach toward creating a new generation of trypanocides.