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，该科包括导致利什曼病、非洲昏睡病和恰加斯病的寄生原虫。剪接先导RNA （slrna）是一种小RNA，通过反式剪接反应为每个核信使RNA提供5'端序列，并且在整个目中在初级序列水平上非常保守。反式剪接是将多顺反子前信使RNA转化为单顺反子信使RNA所必需的，这可能是在着丝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点。本提案概述了在单个蛋白和相互作用复合物水平上详细描述了利什曼原虫和布鲁氏锥虫共有的SL RNA成熟途径的实验，并探讨了SL在翻译中的作用。1)我们将探索cap 4 2'- o -核糖甲基转移酶在布鲁氏菌血流形态中的作用，并利用敲除菌株验证其在前环布鲁氏菌中的功能。我们将研究TbMTr1的帽结合特性，并测试TbMTAP和TbMTr1在核内运输和与假尿嘧啶合成酶复合物相互作用中的作用模型。2)进一步研究参与SL RNA生物发生和功能的复合物。PTP标记、多维蛋白鉴定技术（MudPIT）和布氏体基因组数据库的三位一体使我们能够接近复杂的纯化。着丝质体特异性TbMTr1复合体蛋白TbMTAP参与调控和结构相互作用。利用SmD3蛋白，捕获了SL RNA RNP复合物和相关的剪接机制，包括催化和核心。在鉴定的25种蛋白中，将验证4种新型的着丝质体特异性蛋白与另外两种剪接同源物的关联和功能。这些目标解决了SL RNA成熟过程中决定其后续功能的离散步骤。特异性抑制反式剪接途径对寄生虫是致命的，因此是临床干预的主要目标。公共卫生相关性：动胞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。这些生物采用不寻常的基因表达机制，包括蛋白质编码基因的多顺反子转录和对所有核mrna的通用39个核苷酸剪接先导序列的反式剪接。本研究的目的是继续鉴定剪接的前导特异性加工成分，这些成分有可能成为抗寄生虫化疗的特异性靶点。

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.