GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

拟议研究的长期目标是详细了解 反式剪接在寄生线虫中的生化机制， 蛔虫 从蛔虫胚胎有效制备的无细胞提取物 催化顺式和反式剪接。两种加工反应都需要 几种小核RNA（U snRNA）的参与和条件， 其中体外顺式和/或反式剪接活性可以 取决于添加五种单独的合成物中的任何一种， snRNAs。本文提出的研究将使用该复溶系统， 确定特定U的功能相关序列和结构 snRNAs。特别强调U6和U2 snRNA，因为它们 RNA明确地以催化活性的顺式和反式存在。 剪接体，因为它们可以直接参与催化 拼接提出了五个具体目标： a）确定功能重要的骨架位置（包括 可能的金属配位位点），硫代磷酸酯取代 将采用干扰。B）为了获得一个完整的图片 U6功能所需的核苷酸，碱基特异性化学修饰 将使用干扰分析。（三）功能识别 在U2 snRNA中的重要序列元件，它将受到 扫描阻断诱变。d）阐明剪接位点的机制 反式剪接中的识别和并置，位点特异性 交联（通过在剪接位点或剪接位点附近掺入硫代尿苷） 将被执行。e）为了获得snRNA-snRNA的详细理解， 以及顺式和反式剪接中的snRNA-底物相互作用， 使用短波长紫外线、补骨脂素进行全面的交联分析 并且用硫尿苷的位点或区域特异性取代将被 启动。 结合起来，这些方法可以提供重要的新机制， 深入了解反式剪接并因此阐明其关系 顺式剪接此外，反式剪接是mRNA 在多种医学上重要的人类寄生虫中的成熟，包括 线虫、锥虫和血吸虫。的透彻理解 这种不寻常的RNA加工反应可能提出了新的策略， 对这些寄生虫病进行治疗干预。

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.