Structure and Function of Archaeal and Eukaryotic Box C/D RNPs

古菌和真核 Box C/D RNP 的结构和功能

• 批准号: 0543741
• 负责人: E Stuart Maxwell
• 金额: --
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0543741&HistoricalAwards=false
• 关键词: Structure; Function; Archaeal; Eukaryotic; Box

RNA-guided nucleotide modification complexes direct the post-transcriptional, nucleotide modification of both eukaryotic and archaeal RNAs. The guide RNA of this RNA:protein (RNP) complex base-pairs with the target RNA to determine the specific nucleotide for modification while the RNA-bound proteins catalyze the nucleotide modification reaction. The occurrence of guide RNAs in both Archaea and Eukarya has demonstrated the widespread use of guide RNAs for nucleotide modification and indicated evolutionarily ancient origins for this RNA:protein enzyme. The primary function of the box C/D RNPs is to guide the 2'-O-methylation of nucleotides in target RNAs. Nucleotide methylation is accomplished using both terminal box C/D core and internal C'/D' RNPs contained in the full-length complex. Target RNAs modified by the box C/D RNPs include archaeal and eukaryotic ribosomal RNAs, eukaryotic splicing snRNAs, archaeal tRNAs, and even some eukaryotic messenger RNAs. Previous investigations of the archaeal and eukaryotic box C/D RNPs have defined the essential RNA elements and core proteins required for box C/D RNP assembly and nucleotide methylation. More recently, in vitro box C/D RNP assembly systems have been established using bacterially-expressed recombinant box C/D core proteins. These in vitro systems now provide the opportunity to biochemically dissect the structure and function of these RNP enzymes. Initial investigations have demonstrated the unique structural character of the box C/D and C'/D' RNPs and revealed distinctly different RNP organizations for the archaeal and eukaryotic complexes. Experiments have now revealed that archaeal box C/D RNP assembly requires structural remodeling of the guide RNA with crosstalk interactions between box C/D and C'/D' complexes essential for RNA-guided methylation activity. Proposed experiments will determine how box C/D and C'/D' RNP structural integrity and the inter-RNP spatial distancing of these two complexes affect sRNA remodeling and crosstalk interactions with corresponding effects upon methylation activity. Structural and functional characterization of this minimal archaeal complex will then serve as a model box C/D RNP for analysis of the more structurally complex eukaryotic snoRNP. In vitro and in vivo analysis of eukaryotic box C/D snoRNP will determine if those structural features critical for archaeal sRNP-guided nucleotide methylation are also required for snoRNP-guided methylation activity. Ultimately, comparison of archael and eukaryotic box C/D RNP structure will reveal how gene duplication and altered RNA-binding capabilities of the core proteins has resulted in the evolution of a more structurally complex yet functionally identical eukaryotic snoRNP. This investigation will contribute to the development of human resources in science by continuing to train undergraduate and graduate students as well as postdoctoral fellows. These scientists will learn basic techniques in biochemistry and molecular biology with a strong emphasis on RNA structure and function.

rna引导的核苷酸修饰复合物指导真核生物和古细菌rna的转录后核苷酸修饰。该RNA的向导RNA：蛋白质（RNP）复合物碱基与靶RNA配对，确定要修饰的特定核苷酸，而RNA结合蛋白催化核苷酸修饰反应。古细菌和真核生物中都存在引导RNA，这表明了引导RNA在核苷酸修饰中的广泛使用，并表明了这种RNA的进化古老起源：蛋白质酶。盒状C/D RNPs的主要功能是引导靶rna中核苷酸的2'- o甲基化。核苷酸甲基化是通过末端盒C/D核心和全长复合物中包含的内部C'/D' RNPs来完成的。盒式C/D RNPs修饰的靶rna包括古生菌和真核核糖体rna、真核剪接snrna、古生菌trna，甚至一些真核信使rna。先前对古细菌和真核生物的box C/D RNP的研究已经确定了box C/D RNP组装和核苷酸甲基化所需的基本RNA元件和核心蛋白。最近，利用细菌表达的重组盒C/D核心蛋白建立了体外盒C/D RNP组装系统。这些体外系统现在提供了生化剖析这些RNP酶的结构和功能的机会。初步研究表明，盒状C/D和C'/D' RNP具有独特的结构特征，并揭示了古细菌和真核生物复合物的明显不同的RNP组织。实验表明，古细菌盒C/D RNP组装需要引导RNA的结构重塑，盒C/D和C‘/D’复合物之间的串扰相互作用是RNA引导甲基化活性所必需的。拟议的实验将确定盒C/D和C'/D' RNP结构完整性以及这两个复合物的RNP间空间距离如何影响sRNA重塑和串扰相互作用，并对甲基化活性产生相应的影响。这种最小的古细菌复合体的结构和功能特征将作为模型盒C/D RNP，用于分析结构更复杂的真核生物snoRNP。真核生物盒C/D snoRNP的体内和体外分析将确定古细菌srnp引导的核苷酸甲基化的关键结构特征是否也是snoRNP引导的甲基化活性所必需的。最终，古生物和真核生物盒C/D RNP结构的比较将揭示基因复制和核心蛋白rna结合能力的改变是如何导致真核生物进化出结构更复杂但功能相同的snoRNP的。这项研究将通过继续培养本科生和研究生以及博士后，为科学人力资源的发展做出贡献。这些科学家将学习生物化学和分子生物学的基本技术，重点是RNA的结构和功能。