IN VIVO ANALYSIS OF SL ADDITION IN ASCARIS EMBRYOS

寄生线虫在人类中造成相当大的发病率。淋巴丝虫病、河盲症和钩虫感染了数亿人，仅蛔虫一项就感染了超过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还知之甚少。反式剪接是寄生线虫基因表达的主要机制，占线虫mRNAs表达和成熟的70%以上。剪接前导(SL)反式剪接是一种RNA加工事件，通过将单独转录的小外显子(SL)准确地连接到前mRNAs的5‘端，形成成熟mRNAs的5’末端。寄生线虫中反式剪接的功能意义尚不清楚。我们开发了在蛔虫胚胎中引入和表达核酸的新策略，以便于分析基因表达和反式剪接。随着我们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的表达提供了一个很好的模型。此外，现在第一次有可能解决SL加成在体内的功能意义。通过将荧光素酶报告mRNAs生物导入蛔虫胚胎，我们将检测荧光素酶活性的动力学，以评估翻译效率和功能mRNA的半衰期，以检验几个关于反式剪接的假说，包括：1)前导序列和唯一帽的反式剪接添加在mRNA代谢中是否起重要作用；2)SL添加过程是否起到剪裁mRNAs的作用，去除抑制序列，产生最佳的翻译起始上下文或从mRNA的5‘端到启动子Aug的距离。此外，利用荧光素酶报告和DNA构建，我们将检验寄生线虫中的反式剪接可以起到功能解析多顺反子mRNAs的作用的假设。其他主要目标是研究外显子决定因素对反式剪接效率的作用，并进一步发展直接分析体内RNA加工的生物方法。拟议的研究将解决几个关于反式剪接的突出假设，并提供关于寄生线虫模型中基因表达的主要机制的功能意义的信息。这些研究可能为开发新的、更便宜的治疗药物或候选疫苗提供洞察力，以对抗广泛的寄生虫，包括其他反式剪接生物，如扁虫和动殖体。

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.