Characterization of newly discovered Staufen-mediated RNA decay pathway

新发现的Staufen介导的RNA衰变途径的表征

• 批准号: 7540261
• 负责人: Collynn Fremont Woeller
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7540261
• 关键词: 3' Untranslated Regions; Affect; Arginine; Binding; Bioinformatics; Cell physiology; Complex; Consensus; Degradation Pathway; Dependence; Gene Expression; Gene Expression Regulation; Genes; Goals; Hela Cells; Homeostasis; Human; Laboratories; Mediating; Messenger RNA; Methylation; Mutation; Pathway interactions; Phosphoproteins; Phosphorylation; Physiological; Process; Protein Binding; Protein Kinase; Proteins; RNA Decay; RNA Helicase; RNA Sequences; Research; Role; Site; Structure; Time; Transcript; cell growth; ds RNA-Binding Proteins; mRNA Decay; molecular dynamics

DESCRIPTION (provided by applicant): The goal of this research is to identify the components of the newly discovered Staul-mediated mRNA decay (SMD) pathway so that we will gain a better understanding of its physiological significance and role in post-transcriptional gene regulation. Post-transcriptional gene regulation is increasingly becoming recognized as a crucial aspect of cellular homeostasis. Recently, a new pathway of post-transcriptional gene regulation has been discovered in the Maquat laboratory. The double-stranded RNA binding protein Staufenl (Stau) 1 decreases the levels of up to 1% of all transcripts expressed in human HeLa cells. Staul mediates the degradation of these transcripts in a manner that requires the RNA helicase, Upf1. Notably, Upf1 is an essential component of another mRNA degradation pathway, nonsense-mediated mRNA decay (NMD). Apart from a dependence on Staul and Upf1, little is known about the components or molecular dynamics of SMD. Identifying the other components of SMD and the RNA sequences that bind Staul will increase our understanding of the process. The specific aims are: I. To characterize the complex of proteins that binds the 3' untranslated regions (UTRs) of transcripts targeted for SMD. The complex of proteins that is required for the SMD of several mRNAs will be elucidated and compared. Identifying the proteins involved in SMD will provide important information on the mechanism of SMD. II. To determine the molecular dynamics of Staul-Upf1 complex formation during SMD. Upf1 is a phosphoprotein but the majority of cellular Upf1 is hypophosphorylated. Since Upf1 is activated by phosphorylation during NMD by the protein kinase SMG-1, we will determine if SMG-1 also phosphorylates Upf1 during SMD. Preliminary results suggest Staul is post-translationally modified by arginine methylation. We will determine if Staul is methylated and if methylation affects the ability of Staul to bind Upf1 or otherwise function in SMD. III. To define the RNA sequences/structures that associate with the SMD complex. By generating and analyzing progressive 50-bp deletions and site-directed mutations, and using computational bioinformatics, a consensus SBS will be defined, if there is one. Relevance: Staul has been shown to regulate numerous genes at the mRNA level and has the capacity to be a master regulator of cell growth, division and differentiation. It is important that we elucidate the mechanism whereby Staul and, in particular, SMD regulates gene expression to determine their roles in cellular processes. We believe we are the only group researching this field at the present time.

描述（由申请人提供）： 本研究的目的是鉴定新发现的 Staul 介导的 mRNA 衰减（SMD）途径的组成部分，以便我们更好地了解其生理意义和在转录后基因调控中的作用。转录后基因调控越来越被认为是细胞稳态的一个重要方面。最近，Maquat 实验室发现了一条新的转录后基因调控途径。双链 RNA 结合蛋白 Staufenl (Stau) 1 可降低人类 HeLa 细胞中表达的所有转录本的水平，最高可达 1%。 Staul 以需要 RNA 解旋酶 Upf1 的方式介导这些转录物的降解。值得注意的是，Upf1 是另一种 mRNA 降解途径——无义介导的 mRNA 降解 (NMD) 的重要组成部分。除了对 Staul 和 Upf1 的依赖之外，人们对 SMD 的成分或分子动力学知之甚少。识别 SMD 的其他成分以及结合 Staul 的 RNA 序列将增加我们对该过程的理解。具体目标是： I. 表征结合 SMD 转录物的 3' 非翻译区 (UTR) 的蛋白质复合物。将阐明并比较几种 mRNA 的 SMD 所需的蛋白质复合物。鉴定参与 SMD 的蛋白质将为 SMD 机制提供重要信息。二.确定 SMD 过程中 Staul-Upf1 复合物形成的分子动力学。 Upf1 是一种磷蛋白，但大多数细胞 Upf1 是低磷酸化的。由于 Upf1 在 NMD 期间被蛋白激酶 SMG-1 磷酸化而激活，因此我们将确定 SMG-1 在 SMD 期间是否也会磷酸化 Upf1。初步结果表明 Staul 受到精氨酸甲基化的翻译后修饰。我们将确定 Staul 是否甲基化，以及甲基化是否影响 Staul 结合 Upf1 或在 SMD 中发挥其他功能的能力。三．定义与 SMD 复合物相关的 RNA 序列/结构。通过生成和分析进行性 50 bp 缺失和定点突变，并使用计算生物信息学，将定义共识 SBS（如果有）。相关性：Staul 已被证明可以在 mRNA 水平上调节许多基因，并且有能力成为细胞生长、分裂和分化的主要调节因子。重要的是，我们要阐明 Staul，特别是 SMD 调节基因表达的机制，以确定它们在细胞过程中的作用。我们相信我们是目前唯一研究该领域的团队。