IN VIVO ANALYSIS OF SL ADDITION IN ASCARIS EMBRYOS

蛔虫胚胎中 SL 添加的体内分析

• 批准号: 6615690
• 负责人: RICHARD E. DAVIS
• 金额: $ 26.13万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615690
• 关键词: Ascaris; RNA splicing; gene expression; genetic translation; messenger RNA; microorganism genetics; molecular cloning; nucleic acid metabolism; polymerase chain reaction

Parasitic nematodes cause considerable morbidity in humans. Lymphatic filariasis, river blindness, and hookworms infect hundreds of millions and Ascaris alone infects over 1 billion people. The socioeconomic effects caused by these parasites are severe and present a major obstacle in facilitating medical and economic improvements in many parts of the world. Mechanisms of gene expression in parasitic nematodes are poorly understood. Trans-splicing is a major mechanism of gene expression in parasitic nematodes accounting for greater than or equal to 70 percent of the expression and maturation of nematode mRNAs. Spliced leader (SL) trans-splicing is an RNA processing event that forms the 5' termini of mature mRNAs by accurately joining a small, separately transcribed exon (the SL) to the 5' end of pre- mRNAs. The functional significance of trans-splicing in parasitic nematodes remains unknown. We have developed novel strategies to introduce and express nucleic acids in Ascaris embryos to facilitate analysis of gene expression and trans-splicing. With our development of these molecular genetic tools, Ascaris embryos provide an excellent model for analyzing parasitic nematode gene expression. Moreover, it is now possible for the first time to address the functional significance of SL addition in vivo. Using biolistic introduction of luciferase reporter mRNAs into Ascaris embryos, we will examine the kinetics of luciferase activity to evaluate translational efficiency and functional mRNA half-life to test several hypotheses on trans-splicing including: 1) does trans- splicing addition of a leader sequence and unique cap play an important role in mRNA metabolism and 2) does the process of SL addition serve to trim mRNAs, remove inhibitory sequences, produce an optimal translation initiation context or distance from the 5' end of the mRNA to the initiator AUG. In addition, using luciferase reporters and DNA constructs we will test the hypothesis that trans-splicing in parasitic nematodes can serve to functionally resolve polycistronic mRNAs. Other major goals are to examine the role of exon determinants on trans-splicing efficiency and to further develop biolistic methods to directly analyze RNA processing in vivo. The proposed studies will address several outstanding hypotheses regarding trans-splicing and provide information on the functional significance of a major mechanism of gene expression in a model parasitic nematode. These studies may provide insight into the development of novel and cheaper therapeutic agents or vaccine candidates against a broad spectrum of parasites including other trans-splicing organisms such as flatworms and kinetoplastida.

寄生线虫在人类中引起相当大的发病率。淋巴丝虫病、河盲症和钩虫感染了数亿人，仅蛔虫就感染了超过10亿人。 这些寄生虫造成的社会经济影响是严重的，是促进世界许多地区医疗和经济改善的主要障碍。 寄生线虫的基因表达机制知之甚少。反式剪接是寄生线虫基因表达的主要机制，占线虫mRNA表达和成熟的70%以上。剪接前导序列（SL）反式剪接是通过将小的单独转录的外显子（SL）精确地连接到前mRNA的5'末端而形成成熟mRNA的5'末端的RNA加工事件。 反式剪接在寄生线虫中的功能意义仍然未知。我们已经开发了新的策略，在蛔虫胚胎中引入和表达核酸，以促进基因表达和反式剪接的分析。 随着这些分子遗传学工具的发展，蛔虫胚胎为分析寄生线虫基因表达提供了一个很好的模型。此外，它现在是第一次有可能解决的SL除了在体内的功能意义。 使用将荧光素酶报告基因mRNA生物射弹引入蛔虫胚胎，我们将检查荧光素酶活性的动力学，以评估翻译效率和功能mRNA半衰期，以测试关于反式剪接的几个假设，包括：1）前导序列和独特帽的反式剪接添加在mRNA代谢中是否起重要作用和2）SL添加的过程是否用于修剪mRNA，去除抑制序列，产生最佳的翻译起始环境或从mRNA的5'端到起始物AUC的距离。此外，使用荧光素酶报告基因和DNA构建体，我们将测试寄生线虫中的反式剪接可以用于功能性地解析多顺反子mRNA的假设。 其他主要目标是研究外显子决定簇对反式剪接效率的作用，并进一步开发直接分析体内RNA加工的生物射弹方法。拟议的研究将解决几个突出的假设，反式剪接和提供信息的主要机制的基因表达的功能意义的模型寄生线虫。 这些研究可能会为开发新型且更便宜的治疗剂或候选疫苗提供见解，这些治疗剂或候选疫苗针对广泛的寄生虫，包括其他反式剪接生物，例如扁虫和动质体。