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

描述(申请人提供)：锥虫是血鞭毛类寄生原生动物，在进化早期与其他真核生物有所不同，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。在哺乳动物和昆虫宿主中，确保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)活性的转录启动控制。这种杂乱的转录可能发生在两条DNA链上，对细胞应该是有毒的，特别是对于具有活跃的RNA干扰机制的物种。为了解决这一悖论，我们提出了一个假说，即锥虫体内存在一个核聚腺苷酸化/外切体介导的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来确定不同的非规范聚(A)聚合酶1(NcPAP1)复合体是否标记了特定的RNA类别，以供核外切体降解32-52。我们建议：1)。在体内鉴定ncPAP1的RNA靶点。布氏毛滴虫基因组编码两种典型的核聚(A)聚合酶，它们可能催化mRNA的聚腺苷酸化。在初步研究中，我们已经确定了ncPAP1，这是本应用的主要对象，以及相关的ncPAP2，它是一种对寄生虫非必需的进行性PAP。工作模式是反义的，可能还有其他的转录本，被ncPAP1腺化，从而成为降解的目标。由于我们无法预测ncPAP1的特定靶点，因此将采取两种全球方法来识别体内与ncPAP1结合的RNA并评估转录组的变化。2)。定义ncPAP1的蛋白质相互作用网络。单个ncPAP1多肽在酶活性上是不活跃的，很可能需要一个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解旋酶和第二个可能的死盒解旋酶，但没有外切体成分。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形成一个特定的复合体；2)由ncPAP1作用的转录本通过RNA解旋酶靶向外体。公共卫生相关性：锥虫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，美国对有效的抗锥虫疗法的需求可能是可以预见的。现有的治疗方法往往是有毒和无效的，这就产生了对新药发现的需求。靶向基本的寄生虫特异性酶，例如我们应用中引入的核非正则聚(A)聚合酶，是创造新一代杀锥虫剂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.