Role of Pseudouridylation in Toxoplasma Differentiation

 描述(申请人提供)：修饰的核糖核苷在RNA中含量丰富，但其生物学和结构意义仅在少数情况下为人所知。最丰富的这类核苷是假尿苷，其中尿嘧啶环在转录后“旋转”2个位置，与核糖建立新的连接。尽管在所有被研究的生物体中都存在假尿苷，但只有少数情况下与tRNA和rRNA的结构有关的功能是已知的。最近的结果表明，假尿路也可能发挥重要的调节功能，但它介导这种作用的机制尚不清楚。在这项应用中，描述的工作将首次阐明假尿苷在发育生物学中的作用，使用人类重要的寄生虫弓形虫作为实验系统。刚地弓形虫在感染新宿主后，首先以全身性、快速生长的速殖子的形式扩张，然后分化为生长缓慢、有包囊的缓殖子，从而产生慢性感染。这项工作利用了我们令人惊讶的发现，即假尿苷合成酶(TgPUS1)对弓形虫的无性分化至关重要，从而为确定假尿路在复杂生物过程中的作用提供了机会。提出的第一个目标将使用一种新的方法来基本上识别两种野生型弓形虫的所有假尿路事件。初步结果显示了这种方法的力量，成功地鉴定了许多预期的假尿苷(例如，在rRNA中的保守位置)，以及几种这样的受发育调节的修饰。在目标2中，TgPUS1突变体将被用来确定哪些假尿路事件依赖于这种酶的产生。初步结果再次表明，在几个mRNA中发现了令人惊讶和兴奋的TgPUS1依赖的伪尿苷，证实了该方法的可行性，并证实了TgPUS1的生化活性。目的3将开发一种操纵弓形虫速殖子单个信使核糖核酸上特定假尿嘧啶酸化位点的方法，作为最终测试这种依赖于TgPUS1的假尿嘧啶在速殖子到缓殖子分化中起关键作用的机制的前奏。这里提出的工作的成功执行将为一种重要的人类寄生虫的无性发育提供关键信息，并使人们首次洞察最常见的修饰核糖核苷如何在发育中发挥关键调节作用。

 DESCRIPTION (provided by applicant): Modified ribonucleosides abound in RNA, yet their biological and structural significance are known in only a few cases. The most abundant such nucleoside is pseudouridine in which the uracil ring is post- transcriptionally "rotated" 2 positios to create a new linkage with the ribose. Despite being present in all studied organisms, the function of pseudouridine is known in only a few instances related to the structure of tRNA and rRNA. Recent results suggest that pseudouridylation may also play an important regulatory function but the mechanisms by which it mediates such effects are not known. In this application, work is described that will elucidate, for the first time, the role of pseudouridylatin in developmental biology using the important human parasite, Toxoplasma gondii, as the experimental system. Upon infection of a new host, Toxoplasma gondii initially expands as a systemic, fast-growing tachyzoite, after which it differentiates into a slow-growing, encysted bradyzoite to produce a chronic infection. The work proposed here exploits our surprising discovery that a pseudouridine synthase (TgPUS1) is crucial for the asexual differentiation of Toxoplasma, thereby affording the opportunity to determine the role of pseudouridylation in a complex biological process. The first aim proposed will use a novel method to identify essentially all pseudouridylation events in the two asexual forms of wild type Toxoplasma. Preliminary results show the power of this method with the successful identification of many expected pseudouridines (e.g., in conserved positions in rRNA), as well as several such modifications that are developmentally regulated. In aim 2, TgPUS1 mutants will be used to determine which pseudouridylation events are dependent on this enzyme for their creation. Again, preliminary results show the surprising and exciting discovery of TgPUS1-dependent pseudouridines in several mRNAs establishing the feasibility of the approach and confirming the biochemical activity of the TgPUS1. Aim 3 will develop a method for manipulating specific pseudouridylation sites on individual mRNA species in Toxoplasma tachyzoites as a prelude to eventual testing for the mechanism by which such TgPUS1-dependent pseudouridines play a key role in tachyzoite-to-bradyzoite differentiation. Successful execution of the work proposed here will provide crucial information on the asexual development of an important human parasite as well as giving the first insight into how the most commonly modified ribonucleoside can play a key regulatory role in development.