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茎环结合蛋白（SLBP）如何发挥作用以调节组蛋白mRNA加工和翻译的结构基础。以前的生物化学和遗传学研究已经证明，SLBP是一个最重要的反式作用因子，通过与复制依赖性组蛋白mRNA的3'端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的前mRNA加工、翻译和降解的多蛋白-RNA复合物的募集和组装是重要的。虽然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。我们将采取多学科的方法来结构表征的RNA结合和处理域（RPD）的SLBP，无论是自由和绑定到组蛋白mRNA，使用NMR和质谱。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SLBP如何调节体内组蛋白代谢的模型。本研究的主要目的是：1）确定果蝇SLBP RNA结合和加工结构域的结构和动力学特性（dSLBP RPD）在不存在RNA的情况下使用高分辨率NMR光谱和FT-ICR H/D交换质谱，2）使用高分辨率NMR光谱和FT-ICR H/NMR确定dSLBP RPD茎环组蛋白mRNA复合物的结构和动力学性质。D交换质谱，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物化学性质，4）通过NMR光谱在结构上表征dSLBP RPD突变体，和5）为SLBP对翻译起始因子AD 2和eIF 4G的序列特异性识别提供结构基础。这些研究将为这种生物学重要蛋白质的结构/功能关系提供新的信息，并为长期目标奠定基础，这些目标是（i）了解在组蛋白mRNAs的3'端组装多蛋白复合物的分子决定因素和（ii）开发具有新特异性的RNA结合结构域，可用作生物传感器或细胞生物学研究的试剂。

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.