Kinetoplastid SL RNA Biogenesis

描述（由申请人提供）：我们的研究旨在了解锥虫科的基因表达，该科包括负责利什曼病、非洲昏睡病和恰加斯病的寄生原生动物。重点是剪接前导（SL）RNA的起源和功能，这是一种小RNA，通过反式剪接反应将5 '端序列贡献给每个核信使RNA，并且在整个订单的一级序列水平上非常保守。反式剪接对于将多顺反子前信使RNA转化为单顺反子mRNA是必需的，这可能是动质体中发现的不寻常的基因组组织的结果，并且在人类宿主或昆虫载体中没有发现，因此代表了理想的治疗靶标。该提案概述了实验，详细说明了成熟途径的SL RNA共同的利什曼原虫tarentolae和布氏锥虫在单个蛋白质和相互作用的复合物的水平，并探测SL在翻译中的作用。1)我们将探讨帽4 2 '-O-核糖甲基转移酶在T. brucei，并验证了它们在原循环T.使用敲除菌株的布氏杆菌。我们将研究TbMTr 1的帽结合特性，并测试TbMTAP和TbMTr 1在细胞核中与假尿苷合酶复合物的运输和相互作用中的作用模型。2)进一步表征参与SL RNA生物发生和功能的复合物。PTP标签、多维蛋白质鉴定技术（MudPIT）和T.布鲁氏菌基因组数据库使我们能够接近复杂的纯化。动质体特异性TbMTr 1复合蛋白TbMTAP涉及调节和结构相互作用。使用SmD 3蛋白，SL RNA RNP复合物和相关的剪接机器，催化和核心，已被捕获。在鉴定的25种蛋白质中，将验证4种新的动质体特异性蛋白质与另外两种剪接同源物的关联和功能沿着。这些目标解决了SL RNA成熟中决定其后续功能的离散步骤。反式剪接途径的特异性抑制对寄生虫是致命的，因此是临床干预的主要目标。公共卫生相关性：动质体目含有几种具有医学重要性的病原体，包括人类利什曼病、非洲锥虫病和恰加斯病的病原体。这些生物体采用不寻常的基因表达机制，涉及蛋白质编码基因的多顺反子转录和通用的39个核苷酸的剪接前导序列到所有核mRNA的反式剪接。本研究的目的是继续鉴定有可能成为抗寄生虫化疗特异性靶标的剪接前导序列特异性加工组分。

DESCRIPTION (provided by applicant): Our research is aimed at understanding gene expression in the family Trypanosomatidae, which includes the parasitic protozoa responsible for leishmaniasis, African Sleeping Sickness, and Chagas Disease. The focus is on the genesis and function of the spliced leader (SL) RNA, a small RNA that contributes the 5'-end sequence to every nuclear messenger RNA by a trans-splicing reaction and is conserved extraordinarily at the primary sequence level throughout the Order. Trans-splicing is necessary for the conversion of polycistronic pre-messenger RNA, a likely consequence of the unusual genome organization found in kinetoplastids, into monocistronic mRNA, and is not found in the human host or insect vector, thus representing an ideal therapeutic target. This proposal outlines experiments that detail the maturation pathway of the SL RNA common to Leishmania tarentolae and Trypanosoma brucei at the level of individual proteins and interacting complexes, and probe the role of the SL in translation. 1) We will explore the role of the cap 4 2'-O-ribose methyltransferases in the bloodstream form of T. brucei, and validate their function in procyclic T. brucei using knockout strains. We will investigate the cap-binding properties of TbMTr1, and test models for the role of TbMTAP and TbMTr1 in trafficking and interacting with the pseudouridine synthase complex in the nucleus. 2) To further the characterization of complexes involved in SL RNA biogenesis and function. The trinity of PTP tagging, multidimensional protein identification technology (MudPIT), and the T. brucei genome database enables us to approach complex purification. The kinetoplastid-specific TbMTr1 complex protein TbMTAP is implicated in regulatory and structural interactions. Using the SmD3 protein, the SL RNA RNP complex and associated splicing machinery, both catalytic and core, has been captured. Of the 25 proteins identified, four novel, kinetoplastid-specific proteins will be validated for association and function along with two additional splicing homologs. These goals address discrete steps in the maturation of the SL RNA that determine its subsequent function. Specific inhibition of the trans-splicing pathway is lethal to the parasite and thus a prime target for clinical intervention. PUBLIC HEALTH RELEVANCE: The Order Kinetoplastida contains several pathogens of medical importance including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. These organisms employ unusual mechanisms of gene expression involving polycistronic transcription of protein-coding genes and trans-splicing of a universal 39-nucleotide spliced leader sequence to all nuclear mRNAs. The goal of this study is to continue the identification of spliced leader specific processing components that have the potential to be specific targets for anti-parasite chemotherapy.