Gene Expression in Human Parasitic Nematodes

描述（由申请人提供）：拟议研究的长期目标是详细了解寄生线虫核前mrna剪接的机制，以蛔虫为模型系统。从该生物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的实验系统，其中有可能分析反式剪接使用生化方法。最近的研究已经确定并表征了两种蛋白质因子，SL-30和SL-95，它们是反式剪接所必需的。这些蛋白的功能是促进5‘和3’剪接位点的连接。最近的研究还表明，线虫中的c/s剪接导致成熟mrna上外显子连接络合物的沉积；迄今为止，这种复合物只在哺乳动物细胞中被分析过。这些观察结果可作为未来机制分析的基础，未来机制分析将首先使用耗尽/重构方法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功能的必要条件，而且是充分条件。其次，有证据表明，SL-30与剪接因子SF1/BBP有必要的接触，该蛋白在顺式剪接中起5‘和3’剪接位点之间的桥接作用。耗尽/重构将用于确定SF1/BBP的哪些结构域是顺式和反式剪接所必需的。此外，一个敏感的竞争分析将用于确定SF1/BBP的哪个区域与顺式5'剪接位点的因子通信。最后，同源蛔虫提取物的可用性既催化剪接又有效的体外翻译，为生物化学解决EJC的功能提供了独特的机会。敏感的报告mrna将用于确定EJC是否增强翻译，如果观察到增强，将进行实验以确定增强发生的机制。

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.