GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

人类寄生线虫的基因表达

• 批准号: 6626482
• 负责人: TIMOTHY W. NILSEN
• 金额: $ 42.35万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626482
• 关键词: Ascaris; DNA footprinting; RNA biosynthesis; RNA splicing; X ray crystallography; autoradiography; crosslink; electron microscopy; gene expression; genetic promoter element; messenger RNA; microorganism genetics; nucleic acid hybridization; nucleic acid methylation; nucleic acid sequence; parasitic diseases; posttranscriptional RNA processing; precursor mRNA; site directed mutagenesis; small nuclear RNA; spliceosomes; transcription factor

The long term goal of the proposed research is to achieve a detailed understanding of the mechanism of nuclear pre-mRNA splicing (both cis- and trans-) using cell free extracts derived from embryos of the parasitic nematode, Ascaris. Trans-splicing is used as a mechanism of pre-mRNA processing in a variety of medically relevant parasites, including nematodes, trypanosomatid protaozoa and trematodes; the Ascaris system permits biochemical analysis of this reaction. Four Specific Aims are proposed: 1. Trans-spliceosome assembly involves independent recognition of the 3' splice site on the trans-splice acceptor and the 5' splice site on the SL RNA (the trans-splice donor). A combination of biochemical fractionation and site-specific crosslinking approaches will be used to characterize factors required for 3' splice site recognition. In addition, the hypothesis that the SL RNA is recruited to the acceptor as part of a pre-formed quadruple snRNA will be directly tested.; 2. Accurate maturation of nematode pre-mRNAs requires that the SL be excluded from internal (cis)- 3' splice acceptor sites. Evidence suggests that the cis- 5' splice site and the 5' splice site of the SL RNA are both present and may compete within the cis-spliceosome. Site-directed mutagenesis of both 5' splice sites will be used to determine how a particular 5' splice site is selected for the first transesterification reaction; 3. Tethered (site-specific) hydroxyl radical cleavage will be used to probe the RNA environments of the 5' splice site, the branch point, and the 3' splice site in staged cis- and trans-spliceosomes.; 4. Large (mg) quantities of staged spliceosomes will be purified for the purpose of biophysical analyses; electron cryomicroscopy and X-ray crystallography. In combination, the proposed biochemical and biophysical studies should provide new insight into the mechanism of trans-splicing in particular, and the mechanism of pre-mRNA splicing in general. Finally, a thorough understanding of trans-splicing may suggest possible avenues for therapeutic intervention in diseases caused by parasitic organisms that employ this pre-mRNA processing pathway.

拟议研究的长期目标是使用寄生线虫（Ascaris）胚胎的无细胞提取物详细了解核前mRNA剪接（顺式和反式）的机制。反式剪接被用作多种医学相关寄生虫（包括线虫、锥虫和吸虫）中前体mRNA加工的机制;蛔虫系统允许对该反应进行生化分析。提出了四个具体目标：1。反式剪接体组装涉及反式剪接受体上的3'剪接位点和SL RNA（反式剪接供体）上的5'剪接位点的独立识别。生物化学分级分离和位点特异性交联方法的组合将用于表征3'剪接位点识别所需的因子。此外，SL RNA作为预先形成的四重snRNA的一部分被募集到受体的假设也将被直接检验。2.线虫前体mRNA的准确成熟需要将SL从内部（顺式）-3 '剪接受体位点排除。有证据表明，SL RNA的顺式5'剪接位点和5'剪接位点都存在，并且可能在顺式剪接体内竞争。两个5'剪接位点的定点诱变将用于确定如何选择特定的5'剪接位点用于第一酯交换反应; 3.拴系（位点特异性）羟基自由基切割将用于探测分阶段顺式和反式剪接体中5'剪接位点、分支点和3'剪接位点的RNA环境。4.将纯化大量（mg）分阶段剪接体，用于生物物理分析;电子冷冻显微镜检查和X射线晶体学。结合起来，拟议的生物化学和生物物理学研究应该提供新的见解，特别是反式剪接的机制，和一般的前mRNA剪接的机制。最后，对反式剪接的透彻理解可能为采用这种前mRNA加工途径的寄生生物引起的疾病的治疗干预提供可能的途径。