GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

拟议研究的长期目标是详细了解 寄生线虫反式剪接的生化机制， 蛔虫。高效制备蛔虫胚胎无细胞提取物的研究 催化顺式和反式剪接。这两个处理反应都需要 几种小核RNA(U-SnRNAs)的参与和条件 已经建立了体外顺式和/或反式剪接活性可以 取决于添加五个单独的合成材料中的任何一个 单链RNA。这里提出的研究将使用这个重建系统来 确定特定用途的功能相关序列和结构 单链RNA。我们特别强调U6和U2单链RNA，因为这些 RNA明确地存在于催化活性的顺式和反式- 剪接体，因为它们可能直接参与催化 拼接。提出了五个具体目标： A)确定职能上重要的骨干岗位(包括 可能的金属配位)在U6中，硫代磷酸取代 将使用干扰。B)全面了解 U6功能所需的核苷酸，碱基特异的化学修饰 将使用干涉分析。C)在功能上确定 U2SnRNA中的重要序列元件，它将受到 扫描区块诱变。D)阐明剪接位的作用机制 位点特异性反式剪接中的识别和并列 交联化(通过在剪接部位或其附近掺入硫代尿苷) 将会被执行。E)详细了解SnRNA-SnRNA AS 以及顺式和反式剪接中的SnRNA与底物的相互作用，a 短波紫外光、补骨脂素的综合交联分析 并且特定部位或区域的硫脲取代物将是 已启动。 结合起来，这些方法可能会提供重要的新机制 对反式剪接的洞察，并因此澄清其关系 顺式拼接。此外，反式剪接是mrna的关键步骤。 各种医学上重要的人类寄生虫的成熟，包括 线虫、锥虫和血吸虫。透彻地了解 这种不寻常的RNA加工反应可能会提出新的策略 对这些寄生虫病的治疗干预。

The long term goal of the proposed research is to understand in detail the biochemical mechanism of trans-splicing in the parasitic nematode, Ascaris. Cell free extracts prepared from Ascaris embryos efficiently catalyze cis and trans-splicing. Both processing reactions require the participation of several small nuclear RNAs (U snRNAs) and conditions have been established in which cis and/or trans-splicing activity in vitro can be made dependent upon the addition of any of five individual synthetic snRNAs. The studies proposed here will use this reconstitution system to determine functionally relevant sequences and structures of specific U snRNAs. Particular emphasis is given to U6 and U2 snRNAs because these RNAs are unambiguously present in catalytically active cis and trans- spliceosomes and because they may participate directly in the catalysis of splicing. Five specific aims are proposed: a) To identify functionally important backbone positions (including possible metal-coordination sites) in U6, phosphorothiate substitution interference will be employed. b) To obtain a complete picture of nucleotides required for U6 function, base-specific chemical modification interference analysis will be used. c) To identify functionally significant sequence elements in U2 snRNA, it will be subjected to scanning block-mutagenesis. d) To clarify the mechanism of splice-site recognition and juxtaposition in trans-splicing, site-specific crosslinking (via incorporation of thio uridine at or near splice sites) will be performed. e) To obtain a detailed understanding of snRNA-snRNA as well as snRNA-substrate interactions in cis and trans-splicing, a comprehensive crosslinking analysis using short wave length UV, psoralen and site or region specific substitution with thiouridine will be initiated. In combination, these approaches may provide significant new mechanistic insight into trans-splicing and as a consequence clarify its relationship to cis-splicing. Furthermore, transsplicing is a key step in mRNA maturation in a variety of medically important human parasites including nematodes, trypanosomes, and Schistosomes. A thorough understanding of this unusual RNA processing reaction may suggest novel strategies for therapeutic intervention in these parasitic diseases.