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.
