Phosphorylation dependent recognition of a histone mRNA hairpin by SLBP

SLBP 对组蛋白 mRNA 发夹的磷酸化依赖性识别

• 批准号: 7029023
• 负责人: WILLIAM F. MARZLUFF
• 金额: $ 26.36万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7029023
• 关键词: Drosophilidae; RNA binding protein; gene mutation; genetic manipulation; genetic translation; histones; infrared spectrometry; intermolecular interaction; messenger RNA; nuclear magnetic resonance spectroscopy; phosphorylation; protein biosynthesis; protein degradation; protein isoforms; protein metabolism; protein protein interaction; protein structure function; site directed mutagenesis; transcription factor; translation factor

DESCRIPTION (provided by applicant): The objective of this proposal is to determine the structural basis of how the Stem-Loop Binding Protein (SLBP) functions to regulate histone mRNA processing and translation. Previous biochemical, and genetic studies have demonstrated that SLBP is the single most important trans-acting factor that plays an essential role in histone mRNA metabolism by forming a high affinity complex with a conserved stem-loop at the 3' end of replication-dependent histone mRNAs. The SLBP/RNA complex is important for the recruitment and assembly of multi-protein-RNA complexes that regulate pre-mRNA processing, translation, and degradation of histone mRNAs. Although a wealth of biochemical and genetic information exists for SLBP, the molecular basis for the SLBP-RNA interaction remains to be elucidated. We will take a multi-disciplinary approach to structurally characterize the RNA binding and processing domain (RPD) of SLBP, both free and bound to histone mRNA, using NMR and mass spectrometry. The experiments described in this proposal will complement ongoing functional studies in my laboratory, provide mechanistic information relating to SLBP function, and test currently proposed models as to how SLBP regulates histone metabolism in vivo. The specific aims of the proposal are 1) to determine the structural and dynamic properties of the Drosophila SLBP RNA binding and processing domain (dSLBP RPD) in the absence of RNA using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 2) to determine the structural and dynamic properties of the dSLBP RPD stem-loop histone mRNA complex using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 3) to characterize the biological and biochemical properties of sSLBP RPD mutants impaired in RNA binding and RNA processing, 4) to structurally characterize dSLBP RPD mutants by NMR Spectroscopy, and 5) to provide a structural basis for sequence specific recognition of the translation initiation factors AD2 and elF4G by SLBP. These studies will provide new information on structure/function relationships for this biological important protein and also lay the groundwork for long-term goals which are (i) to understand the molecular determinants for assembly of multi-protein complexes at the 3' end of histone mRNAs and (ii) to develop RNA binding domains with novel specificities that can be used as biosensors or reagents for cell biological studies.

描述（由申请人提供）：本提案的目的是确定干环结合蛋白（SLBP）如何发挥调节组蛋白 mRNA 加工和翻译功能的结构基础。先前的生化和遗传学研究表明，SLBP 是最重要的单一反式作用因子，通过与复制依赖性组蛋白 mRNA 3' 端的保守茎环形成高亲和力复合物，在组蛋白 mRNA 代谢中发挥重要作用。 SLBP/RNA 复合物对于调节组蛋白 mRNA 前体 mRNA 加工、翻译和降解的多蛋白-RNA 复合物的募集和组装非常重要。尽管 SLBP 存在丰富的生化和遗传信息，但 SLBP-RNA 相互作用的分子基础仍有待阐明。我们将采用多学科方法，使用 NMR 和质谱法对 SLBP 的 RNA 结合和处理结构域 (RPD)（游离的和与组蛋白 mRNA 结合的结构域）进行结构表征。本提案中描述的实验将补充我实验室正在进行的功能研究，提供与 SLBP 功能相关的机制信息，并测试目前提出的关于 SLBP 如何调节体内组蛋白代谢的模型。该提案的具体目标是 1) 在没有 RNA 的情况下，使用高分辨率 NMR 光谱和 FT-ICR H/D 交换质谱法确定果蝇 SLBP RNA 结合和处理结构域 (dSLBP RPD) 的结构和动态特性，2) 使用高分辨力确定 dSLBP RPD 茎环组蛋白 mRNA 复合物的结构和动态特性。 分辨率 NMR 光谱和 FT-ICR H/D 交换质谱，3) 表征 RNA 结合和 RNA 加工受损的 sSLBP RPD 突变体的生物学和生化特性，4) 通过 NMR 光谱对 dSLBP RPD 突变体进行结构表征，5) 为翻译起始因子 AD2 和 AD2 的序列特异性识别提供结构基础 SLBP 的 eF4G。这些研究将为这种重要的生物学蛋白质的结构/功能关系提供新的信息，并为长期目标奠定基础，这些目标是：(i) 了解组蛋白 mRNA 3' 端多蛋白复合物组装的分子决定因素，以及 (ii) 开发具有新颖特异性的 RNA 结合结构域，可用作细胞生物学研究的生物传感器或试剂。