Kinetoplastid SL RNA biogenesis

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。反式剪接是将多顺反子前信使RNA(可能是动植体中发现的不寻常基因组组织的结果)转化为单顺反子mRNA所必需的，但在人类宿主或昆虫载体中没有发现，因此代表了一个可能的治疗靶点。这项提案概述了三组互补的实验，详细说明了塔伦托利什曼原虫和布氏锥虫共同的SL RNA的成熟途径。1)完善准确有效的成熟和反式剪接所需的顺式元件(SL RNA序列和结构)的图谱。所获得的数据将使未来能够识别与RNA重要区域相互作用的反式因子(蛋白质和RNA)。2)分离和研究依赖XPO1的核出口复合体在SL RNA运输中的作用。3)一个3‘-核酸外切酶和两个核糖2’-O-甲基转移酶在SL RNA 3‘和5’端成熟过程中的作用的实验研究，以及这些蛋白质与SL RNA相互作用的生物信息学鉴定和模拟。这三个目标解决了SL RNA成熟过程中的离散步骤，这些步骤决定了其后续功能。反式剪接途径的特异性抑制对细胞是致命的，因此是抗寄生虫干预的主要目标。

DESCRIPTION (provided by the applicant): Our research is aimed at understanding gene expression in the family Trypanosomatidae, which includes the parasitic protozoon's 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 via a trans-splicing reaction. Trans-splicing, which is necessary for the conversion of polycistronic pre-messenger RNA (a likely consequence of the unusual genome organization found in kinetoplastids) into monocistronic mRNA, is not found in the human host or insect vector and thus represents a possible therapeutic target. This proposal outlines three complementary sets of experiments that detail the maturation pathway of the SL RNA common to Leishmania tarentolae and Trypanosoma brucei. 1) To refine the map of cis elements (SL RNA sequences and structures) necessary for accurate and efficient maturation and trans-splicing. Data obtained will allow the future identification of trans factors (protein and RNA) that interact with essential regions of RNA. 2) To isolate and examine the role of an XPO1-dependent nuclear export complex in SL RNA trafficking. 3) Experimental examination of the roles of a 3'-exonuclease and two ribose 2'-O- methyltransferases in the maturation of the 3' and 5' ends of the SL RNA, and bioinformatic identification and modeling of these proteins' interaction with the SL RNA. These three 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 cell and thus a prime target for anti-parasite intervention.