Phosphorylation dependent recognition of a histone mRNA hairpin by SLBP

描述(由申请人提供)：本提案的目的是确定干环结合蛋白(SLBP)如何发挥功能来调节组蛋白mRNA的加工和翻译的结构基础。以往的生化和遗传学研究表明，SLBP是唯一最重要的反式作用因子，通过在复制依赖的组蛋白mRNAs的3‘端与保守的茎环形成高亲和力的复合体，在组蛋白mRNA的代谢中发挥重要作用。SLBP/RNA复合体对于多蛋白-RNA复合体的招募和组装是重要的，这些复合体调节组蛋白mRNAs的前mRNA的加工、翻译和降解。尽管SLBP存在着丰富的生化和遗传信息，但SLBP与RNA相互作用的分子基础仍有待阐明。我们将采取多学科的方法，利用核磁共振和质谱仪对SLBP的RNA结合和加工结构域(RPD)进行结构表征，包括游离的和与组蛋白mRNA结合的。这项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如何在体内调节组蛋白代谢的模型。该建议的具体目的是1)使用高分辨率核磁共振光谱和FT-ICR H/D交换质谱仪确定没有RNA的果蝇SLBP RNA结合和加工结构域(DSLBP RPD)的结构和动力学性质，2)使用高分辨率核磁共振光谱和FT-ICR H/D交换质谱学确定dSLBP RPD茎环组蛋白mRNA复合体的结构和动力学性质，3)表征在RNA结合和RNA加工中受损的sSLBP RPD突变体的生物学和生化性质，4)通过核磁共振光谱对dSLBP RPD突变体进行结构表征，5)为SLBP序列特异性识别翻译起始因子AD2和elF4G提供结构基础。这些研究将为这种生物重要蛋白质的结构/功能关系提供新的信息，并为长期目标奠定基础，这些长期目标是：(I)了解组蛋白mRNAs 3‘端多蛋白复合体组装的分子决定因素；(Ii)开发具有新特性的RNA结合域，可用作细胞生物学研究的生物传感器或试剂。

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.