Kinetoplastid SL RNA Biogenesis

动质体 SL RNA 生物发生

• 批准号: 8284373
• 负责人: DAVID A CAMPBELL
• 金额: $ 37.24万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8284373
• 关键词: Address; African Trypanosomiasis; Binding; Biogenesis; Biology; Blood Circulation; Catalytic Domain; Cell Line; Cell Nucleolus; Cell Nucleus; Chagas Disease; Clinical; Code; Complement; Complex; Core Protein; Disease; Enzymes; Exons; Exonuclease; Family; Family member; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; Health; Homologous Gene; Human; Hypermethylation; In Vitro; Individual; Insect Vectors; Intervention; Kinetoplastida; Knock-out; Leishmania; Leishmaniasis; Life; Link; Medical; Messenger RNA; Methylation; Methyltransferase; Mitochondrial RNA; Modification; Molecular; Names; Nuclear; Nucleotides; Organism; Parasites; Pathway interactions; Play; Polishes; Positioning Attribute; Process; Property; Protein Splicing; Proteins; Protozoa; Pseudouridine; RNA Processing; RNA Splicing; Reaction; Research; Ribonucleoproteins; Ribose; Ribosomal RNA; Role; Route; Small Nuclear RNA; Small RNA; Spliced Leader RNA; Spliced Leader Sequences; Staging; Technology; Testing; Therapeutic; Trans-Splicing; Transfer RNA; Translations; Trypanosoma brucei brucei; Trypanosomatina; Uridine; Validation; Work; chemotherapy; combat; fight against; genome database; in vivo; insertion/deletion mutation; insight; interest; novel; particle; pathogen; protein complex; protein purification; research study; role model; spleen exonuclease; therapeutic target; trafficking; vector

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.

描述(申请人提供)：我们的研究旨在了解锥虫科的基因表达，包括引起利什曼病、非洲睡眠病和恰加斯病的寄生原虫。重点是剪接前导(SL)RNA的起源和功能，它是一种小RNA，通过反式剪接反应将5‘端序列贡献给每个核信使RNA，并在整个目的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RNA所必需的，这可能是动植体中发现的不寻常基因组结构的结果，但在人类宿主或昆虫载体中没有发现，因此是理想的治疗靶点。这项建议概述了实验，详细描述了利什曼原虫和布氏锥虫共同的SL RNA在单个蛋白质和相互作用复合体水平上的成熟途径，并探讨了SL在翻译中的作用。1)我们将探索4‘-O-核糖甲基转移酶在布鲁氏毛滴虫血流中的作用，并利用基因敲除株验证它们在原环状毛滴虫中的功能。我们将研究TbMTr1的帽子结合性质，并测试TbMTAP和TbMTr1在运输和与细胞核中的伪尿苷合成酶复合体相互作用中的作用模型。2)进一步研究与SL RNA生物发生和功能有关的复合体。三位一体的PTP标签、多维蛋白质鉴定技术(MudPIT)和布鲁氏锥虫基因组数据库使我们能够接近复杂的纯化方法。动质体特异性TbMTr1复合蛋白TbMTAP参与调控和结构相互作用。利用SmD3蛋白，已经捕获了SL RNA RNP复合体和相关的剪接机制，包括催化和核心。在已确定的25个蛋白质中，四个新的动质体特异性蛋白质将与另外两个剪接同源物一起被验证关联和功能。这些目标解决了SL RNA成熟过程中的离散步骤，这些步骤决定了其后续功能。对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。与公共卫生相关：动塑形目包含几种具有重要医学意义的病原体，包括人类利什曼病、非洲锥虫病和恰加斯病的病原体。这些生物使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所有核mRNAs的通用39核苷酸剪接前导序列的反式剪接。本研究的目标是继续鉴定有可能成为抗寄生虫化疗特异性靶点的剪接前导的特定加工成分。