Gene Expression in Human Parasitic Nematodes

人类寄生线虫的基因表达

• 批准号: 7025810
• 负责人: TIMOTHY W. NILSEN
• 金额: $ 44.68万
• 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/7025810
• 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以蛔虫为模型系统详细了解寄生线虫中核前mRNA剪接的机制。从该生物体发育中的胚胎制备的无细胞提取物有效地催化顺式和反式剪接；这些提取物仍然是唯一可以使用生化方法分析反式剪接的实验系统。最近的研究已经确定并鉴定了两个蛋白质因子，SL-30和SL-95，它们是反式剪接所唯一需要的。这些蛋白质的功能是促进5‘和3’剪接位点的连接。最近的研究还表明，线虫中的c/S剪接导致外显子连接复合体成熟mRNAs的沉积；到目前为止，这种复合体只在哺乳动物细胞中被分析过。这些观察结果将作为未来机制分析的基础，这将首先使用耗尽/重建的方法来进一步研究反式剪接特定因子的功能。此外，将采用融合蛋白策略来确定SL-30和SL-95是否不仅是SL RNP功能所必需的，而且是充分发挥作用的。第二，证据表明SL-30与剪接因子SF1/BBP进行必要的接触，SF1/BBP是一种在顺式剪接中起5‘和3’剪接位点之间桥梁作用的蛋白质。耗尽/重建将用于确定顺式和反式剪接都需要哪些SF1/BBP结构域。此外，敏感的竞争分析将被用来确定SF1/BBP的哪个区域与顺式5‘剪接位点处的因子通信。最后，可催化剪接和高效体外翻译的同源蛔虫提取物的可获得性为从生化角度解决EJC的功能提供了独特的机会。敏感的报告mRNA将被用来确定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。 总而言之，拟议的研究应该为特别是反式剪接和一般剪接的机制提供新的见解。它们还可能提供与剪接和后处理mRNA代谢的耦合相关的新信息。