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

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：蛋白质（RNP）复合物的指导RNA与靶RNA碱基配对以确定用于修饰的特定核苷酸，而RNA结合的蛋白质催化核苷酸修饰反应。 指导RNA在真核生物和真核生物中的出现证明了指导RNA在核苷酸修饰中的广泛应用，并表明了这种RNA：蛋白酶的进化古老起源。 盒C/D RNP的主要功能是引导靶RNA中核苷酸的2 '-O-甲基化。 核苷酸甲基化使用全长复合物中包含的末端盒C/D核心和内部C '/D' RNP两者来完成。 由盒C/D RNP修饰的靶RNA包括古细菌和真核生物核糖体RNA、真核生物剪接snRNA、古细菌tRNA，甚至一些真核生物信使RNA。 先前对古细菌和真核生物盒C/D RNP的研究已经确定了盒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，已经使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机会，以生化解剖这些RNP酶的结构和功能。 初步的研究已经证明了盒C/D和C '/D' RNP的独特结构特征，并揭示了古细菌和真核生物复合物的明显不同的RNP组织。 实验现已揭示，古细菌盒C/D RNP组装需要引导RNA的结构重构，其中盒C/D和C '/D'复合物之间的串扰相互作用对于RNA引导的甲基化活性是必需的。提出的实验将确定盒C/D和C '/D' RNP结构完整性和这两种复合物的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。 这种最小的古菌复合物的结构和功能表征，然后将作为一个模型盒C/D RNP的结构更复杂的真核snoRNP的分析。 真核盒C/D snoRNP的体外和体内分析将确定古细菌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需的。 最终，古细菌和真核细胞盒C/D RNP结构的比较将揭示基因复制和改变核心蛋白的RNA结合能力如何导致结构更复杂但功能相同的真核snoRNP的进化。这项调查将有助于科学人力资源的发展，继续培养本科生和研究生以及博士后研究员。 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。

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.