Gene Expression in Human Parasitic Nematodes

人类寄生线虫的基因表达

• 批准号: 6879483
• 负责人: TIMOTHY W. NILSEN
• 金额: $ 44.42万
• 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 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879483
• 关键词: Ascaris; RNA biosynthesis; RNA splicing; cell free system; chimeric proteins; gene expression; genes; genetic translation; helminth genetics; microorganism genetics; precursor mRNA; protein protein interaction; protein structure; small nuclear RNA; small nuclear ribonucleoproteins; transcription factor

DESCRIPTION (provided by the applicant): The broad long term goal of the proposed research is to understand in detail the mechanism of nuclear pre-mRNA splicing in parasitic nematodes using Ascaris as a model system. Cell free extracts prepared from developing embryos of this organism efficiently catalyze both cis and trans-splicing; these extracts remain the only experimental system in which it is possible to analyze trans-splicing using biochemical approaches. Recent studies have identified and characterized two protein factors, SL-30 and SL-95 that are uniquely required for trans-splicing. These proteins function to promote joining of the 5' and 3' splice sites. Recent studies have also revealed that c/s-splicing in nematodes results in the deposition of an exon junction complexon mature mRNAs; to date this complex has only been analyzed in mammalian cells. These observations serve as the basis for future mechanistic analyses, which will first use a depletion/reconstitution approach to study further the function of the trans-splicing specific factors. In addition, fusion protein strategies will be employed to determine if the SL-30 and SL-95 are not only necessary but sufficient for SL RNP function. Second, the evidence indicates that SL-30 makes essential contacts with the splicing factor SF1/BBP, a protein that plays a bridging role between 5' and 3' splice sites in cis-splicing. Depletion/reconstitution will be used to determine which domains of SF1/BBP are required for both cis and trans-splicing. In addition, a sensitive competition assay will be used to determine which region of SF1/BBP communicates with factors at cis 5' splice sites. Finally, the availability of homologous Ascaris extracts that catalyze both splicing and efficient in vitro translation provides a unique opportunity to biochemically address the function of the EJC. Sensitive reporter mRNAs will be used to determine if the EJC enhances translation and, if enhancement is observed, experiments will be conducted to determine the mechanism by which enhancement occurs. In combination, the proposed studies should provide new insight into the mechanism of trans-splicing in particular and splicing in general. They may also provide novel information relevant to the coupling of splicing and post-processing mRNA metabolism.

描述（由申请人提供）：拟议研究的广泛长期目标是详细了解使用Ascaris作为模型系统的寄生线虫中核MRNA剪接的机制。通过开发这种生物的胚胎制备的无细胞提取物有效地催化顺式和变形。这些提取物仍然是唯一可以使用生化方法来分析跨拼接的实验系统。最近的研究已经确定并表征了两个蛋白质因子SL-30和SL-95，这些因子是跨拼接所需的。这些蛋白质的功能可促进5'和3'剪接位点的连接。最近的研究还表明，线虫中的c/s-splics s-splicing导致外显子连接复合物成熟mRNA的沉积。迄今为止，该复合物仅在哺乳动物细胞中进行了分析。这些观察结果是未来机械分析的基础，该分析将首先使用耗竭/重构方法来进一步研究移层特定因素的功能。另外，将采用融合蛋白策略来确定SL-30和SL-95是否不仅需要SL-30和SL-95。其次，证据表明SL-30与剪接因子SF1/BBP是必不可少的，该蛋白质在顺式拼写中起着5'和3'的剪接位点起着桥接作用的蛋白质。耗竭/重构将用于确定顺式和移媒体所需的SF1/BBP的域。此外，将使用敏感的竞争测定法来确定SF1/BBP的哪个区域与CIS 5'剪接位点的因素进行通信。最后，催化剪接和有效的体外翻译的同源as虫提取物的可用性为生化解决了EJC的功能提供了独特的机会。敏感的记者mRNA将用于确定EJC是否增强了翻译，如果观察到增强，将进行实验，以确定发生增强的机制。 结合起来，拟议的研究应提供新的洞察力，尤其是跨剪接的机制，尤其是剪接。他们还可以提供与剪接和后加工mRNA代谢的耦合有关的新颖信息。