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）：修饰的核糖核苷在RNA中大量存在，但它们的生物学和结构意义仅在少数情况下已知。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位以与核糖产生新的连接。尽管存在于所有研究的生物体中，但假尿苷的功能仅在与tRNA和rRNA结构相关的少数情况下已知。最近的研究结果表明，假尿苷化也可能发挥重要的调节功能，但它介导这种影响的机制尚不清楚。在这个应用程序中，工作描述，将阐明，第一次，pseudouridylatin在发育生物学中的作用，使用重要的人类寄生虫，弓形虫，作为实验系统。刚地弓形虫感染新的宿主后，最初以快速生长的速殖子形式全身扩张，之后分化为缓慢生长的包囊缓殖子，从而产生慢性感染。这里提出的工作利用了我们令人惊讶的发现，假尿苷合酶（TgPUS1）是弓形虫无性分化的关键，从而提供了机会，以确定假尿苷化在一个复杂的生物过程中的作用。提出的第一个目标将使用新的方法来鉴定野生型弓形虫的两种无性形式中基本上所有的假尿苷化事件。初步结果显示了该方法的能力，成功鉴定了许多预期的假尿苷（例如，在rRNA的保守位置），以及几个这样的修饰，发育调控。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该酶来产生。同样，初步结果显示了在几种mRNA中TgPUS1依赖性假尿苷的令人惊讶和令人兴奋的发现，建立了该方法的可行性并证实了TgPUS1的生物化学活性。目的3将开发一种方法来操纵特定的pseudouridylation位点的个别mRNA物种在弓形虫速殖子的前奏，最终测试的机制，这种TgPUS1依赖性pseudouridines发挥关键作用，在速殖子到缓殖子分化。成功地执行这里提出的工作将提供重要的人类寄生虫的无性发育的关键信息，以及给最常见的修饰核糖核苷如何在发展中发挥关键的调节作用的第一个见解。