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（由申请人提供）：RNA中富含修饰的核糖核苷，但仅在少数情况下已知它们的生物学和结构意义。最丰富的此类核苷是假尿苷，其中尿嘧啶环在转录后“旋转”2个位置以产生。尽管假尿苷存在于所有研究的生物体中，但其功能仅在少数与 tRNA 和 tRNA 结构相关的情况下为人所知。最近的结果表明，假尿苷化也可能发挥重要的调节功能，但其介导这种作用的机制尚不清楚。在本申请中，描述了将首次阐明假尿苷化在发育生物学中的作用的工作。使用重要的人类寄生虫弓形虫作为实验系统，在感染新宿主后，弓形虫最初会扩展为全身性、快速生长的速殖子，然后分化。转化为缓慢生长的包囊缓殖子以产生慢性感染，本文提出的工作利用了我们令人惊讶的发现，即假尿苷合酶（TgPUS1）对于弓形虫的无性分化至关重要，从而提供了确定假尿苷化在弓形虫无性分化中的作用的机会。提出的第一个目标是使用一种新方法来识别野生型弓形虫的两种无性形式中的所有假尿苷化事件，初步结果显示了其威力。该方法成功鉴定了许多预期的假尿苷（例如，在 rRNA 中的保守位置），以及发育调控的几种此类修饰。在目标 2 中，TgPUS1 突变体将用于确定哪些假尿苷化事件依赖于此。再次，初步结果显示在几种 mRNA 中发现了 TgPUS1 依赖性假尿苷，这证明了该方法的可行性并证实了该方法的生化活性。 TgPUS1。目标 3 将开发一种操作弓形虫速殖子中单个 mRNA 物种上的特定假尿苷化位点的方法，作为最终测试此类 TgPUS1 依赖性假尿苷在速殖子到缓殖子分化中发挥关键作用的机制的前奏。这里提出的工作将提供有关重要人类寄生虫无性发育的重要信息，并首次深入了解最常见的修改方式核糖核苷在发育中可以发挥关键的调节作用。