Role of Pseudouridylation in Toxoplasma Differentiation

假尿苷化在弓形虫分化中的作用

• 批准号: 8981871
• 负责人: John C Boothroyd
• 金额: $ 24.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981871
• 关键词: Active Sites; Area; Biochemical; Bioinformatics; Biological; Biological Process; Biology; Cells; Chemical Modifier; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complementary DNA; Complex; Development; Developmental Biology; Disease; Engineering; Enzymes; Event; Future; Genes; Goals; Human; In Vitro; Individual; Infection; Insertional Mutagenesis; Knock-out; Mediating; Messenger RNA; Methods; Modification; Nucleic Acids; Nucleosides; Organism; Parasites; Phenocopy; Play; Positioning Attribute; Preparation; Primer Extension; Process; Property; Pseudouridine; RNA; RNA-Directed DNA Polymerase; Research; Ribonucleosides; Ribose; Ribosomal RNA; Role; Sampling; Site; Small Nucleolar RNA; Staging; Structure; System; Testing; Time; Toxoplasma; Toxoplasma gondii; Transfer RNA; Untranslated RNA; Uracil; Work; asexual; base; deep sequencing; enzyme activity; insight; mutant; novel; prevent; public health relevance; success; tool

 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.

 描述（适用提供）：RNA中的修饰的核核​​苷比比皆是，但它们的生物学和结构意义仅在少数情况下才知道。最丰富的核外侧是伪嘧啶，其中乌拉西尔环在转录后“旋转” 2个位置，以与结核孔建立新的联系。尽管存在于所有Studiod生物中，但仅在与tRNA和rRNA的结构有关的少数情况下，伪氨酸的功能是众所周知的。最近的结果表明，假基因化也可能起重要的调节功能，但是介导这种作用的机制尚不清楚。在此应用中，描述的工作将首次阐明使用重要人类寄生虫Toxoplasma gondii作为实验系统，将伪基丁蛋白在发育生物学中的作用。在感染新宿主后，弓形虫Gondii最初会作为一种系统性，快速增长的tachyzoite扩展，之后，它将其区分为生长缓慢的，经过良好的牛皮岩，以产生慢性感染。此处提出的工作利用了我们惊讶的发现，即假氨酸合酶（TGPUS1）对于弓形虫的无性区分至关重要，从而有机会确定假羟基化在复杂的生物学过程中的作用。提出的第一个目的将使用一种新颖的方法来识别两种无性野生型弓形虫中的所有假胞苷化事件。初步结果表明，通过成功识别许多预期的假蛋白（例如，在RRNA中配置的位置）以及几种经过调节的修改。在AIM 2中，将使用TGPUS1突变体来确定哪些假基因化事件取决于该酶的创造。同样，初步结果表明，在几个mRNA中，TGPUS1依赖性假氨酸的令人惊讶而令人兴奋的发现，这些mRNA建立了该方法的可行性并确认了TGPUS1的生化活性。 AIM 3将开发一种方法，用于在弓形虫tachyzoites中各个mRNA物种上操纵特定的假性位点，以此作为事件测试的序幕，以使这种TGPUS1依赖性假源在tachyzoite-tachyzoite-to-bradyzoite差异化中起着关键作用。在这里提出的工作的成功执行将提供有关人类寄生虫的无性发展的重要信息，并首先了解最常见的修饰色带视觉如何在开发中起关键的调节作用。