The Biochemistry and Biology of C. elegans Poly(U) Polymerases

线虫聚 (U) 聚合酶的生物化学和生物学

• 批准号: 8594518
• 负责人: Melanie A. Preston
• 金额: $ 5.39万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8594518
• 关键词: Address; Animals; Behavior; Biochemical; Biochemistry; Biological; Biological Process; Biology; C. elegans genome; Caenorhabditis elegans; Cell physiology; Cells; Development; Diabetes Mellitus; Diagnostic; Disease; Enzymes; Family; Fission Yeast; Foundations; Future; Gene Expression; Generations; Genetic; Goals; Growth; Health; Human; Immune; Immune response; Investigation; Learning; Length; Link; Messenger RNA; Metabolism; MicroRNAs; Modeling; Molecular Genetics; Myotonic Dystrophy; Nematoda; Nuclear; Nuclear RNA; Pathway interactions; Phenotype; Physiology; Polyadenylation; Polynucleotide Adenylyltransferase; Proteins; RNA; RNA Interference; Reading; Regulation; Role; Small RNA; Solid; Study models; System; Tail; Time; Tissues; Transferase; Transgenes; Uridine; Work; Yeasts; cancer stem cell; genome-wide; in vitro activity; in vivo; insight; mRNA Transcript Degradation; poly U polymerase; positional cloning; pre-miRNA; public health relevance; research study; stem cells; tool; tumorigenesis

DESCRIPTION (provided by applicant): The goal of this proposal is to establish a foundation for understanding the roles of poly(U) polymerases (PUPs) and RNA uridylation. PUPs are conserved from fission yeast to humans and add uridines to the 3' end of RNA substrates to form U tails1-10. PUPs act on an exceptionally broad range of substrates, from microRNAs (miRNAs) to mRNAs, and the effects of U tails are astoundingly diverse. U tails can cause degradation9,14-17, stabilization18,19, altered function6, altered processing4-6,20, altered translation15,21,22, and mRNA editing10. Investigation of the roles of PUPs impacts human health. PUPs and several of their RNA substrates have been directly linked to tumorigenesis11, proliferation5,8,12, stem cell maintenance4, and immune response6,13. I will use C. elegans as model to begin to understand the biological roles of PUPs and of U tails. The experiments in this proposal will identify C. elegans PUPs and then develop both biochemical tools to identify RNA substrates and genetic tools to assess PUP biological functions. Aim 1: Identify C. elegans PUPs and begin to elucidate their biochemical and biological activities. PUPs belong to the family of beta-nucleotidyl transferases that act on RNA (rNTRs), which includes noncanonical poly(A) polymerases (PAPs)36. PUPs cannot be distinguished from noncanonical PAPs by sequence similarity alone. The C. elegans genome encodes six additional noncanonical rNTRs, as determined by sequence similarity to known PUPs and noncanonical PAPs. Experiments in Aim 1 will identify C. elegans PUPs and then further examine PUP activity in vitro. Genetic studies in Aim 1 will begin to address the functions of PUP-2, PUP-3, and additional germline PUPs by using diagnostic phenotypes to assess the functional crosstalk between PUPs in one tissue. Aim 2: Identify RNA substrates of PUP-2 and of PUP-3 on a genome-wide scale. PUPs can act on diverse RNAs and elicit a variety of effects. Uridylation triggers degradation of mRNAs and siRNAs14,15,21, but stabilizes U6 small nuclear RNA18,19. The lengths of U tails required for such effects can be astonishingly short: 1-3 U's inhibits miR-26 function6. Identification of the substrates of PUPs is key to understanding the importance of uridylation and may also link PUPs to known biological processes, much as Lin28-dependent TUT4 activity on pre-miRNA links PUP activity to development, tumorigenesis, and other disease states4,11,37-39. The experiments in Aim 2 will be the first global analyses of the RNA substrates of PUPs. They will likely reveal new classes of RNAs subjected to PUP control and provide insight into PUP biological roles. The field is still in its early days of understanding how PUPs work, on which RNAs they act, and their biological significance. Yet already, it is clear that PUPs have key roles in a wide variety of disease states. Our studies will provide a strong molecular and genetic foundation for understanding the roles of PUPs in humans.

描述（由申请人提供）：本提案的目标是为理解聚(U)聚合酶（PUPs）和RNA尿苷化的作用建立基础。幼崽从裂变酵母到人类是保守的，并在RNA底物的3'端添加尿苷，形成U尾s1-10。pup作用于非常广泛的底物，从microRNAs （miRNAs）到mrna， U尾的作用是惊人的多样化。U尾可导致降解9,14-17、稳定18,19、功能改变6、加工改变4-6,20、翻译改变15、21、22和mRNA编辑10。pup对人体健康影响作用的研究。pup及其几种RNA底物与肿瘤发生、增殖、干细胞维持和免疫反应直接相关。我将以秀丽隐杆线虫为模型，开始了解pup和U尾的生物学作用。本实验将鉴定秀丽隐杆线虫的PUP，然后开发生化工具来鉴定RNA底物和遗传工具来评估PUP的生物学功能。目的1：鉴定秀丽隐杆线虫幼崽，并开始阐明其生化和生物学活性。PUPs属于作用于RNA （rNTRs）的β -核苷酸转移酶家族，其中包括非规范聚(A)聚合酶（pap）36。pup不能仅通过序列相似性来区分非规范pap。秀丽隐杆线虫基因组编码6个额外的非规范rNTRs，这是由已知pup和非规范pap的序列相似性决定的。Aim 1的实验将鉴定秀丽隐杆线虫的PUP，然后进一步检测PUP的体外活性。Aim 1中的遗传学研究将通过使用诊断表型来评估一个组织中pup之间的功能串扰，开始解决PUP-2， PUP-3和其他种系pup的功能。目的2：在全基因组范围内鉴定PUP-2和PUP-3的RNA底物。pup可以作用于多种rna，并引发多种效应。尿苷化触发mrna和sirna降解14,15,21，但稳定U6小核rna18,19。这种作用所需的U尾长度可能短得惊人：1-3 U尾可以抑制miR-26的功能6。鉴定PUP的底物是理解尿苷化重要性的关键，也可能将PUP与已知的生物学过程联系起来，就像lin28依赖性的前mirna上的TUT4活性将PUP活性与发育、肿瘤发生和其他疾病状态联系起来一样4,11,37-39。Aim 2中的实验将是首次对pup的RNA底物进行全面分析。他们可能会揭示受PUP控制的新rna类别，并提供对PUP生物学作用的见解。在了解pup的工作原理、作用于哪些rna以及它们的生物学意义方面，该领域仍处于早期阶段。然而，已经很清楚的是，pup在多种疾病状态中起着关键作用。我们的研究将为理解pup在人类中的作用提供强有力的分子和遗传学基础。