Structural Features of the Guide:Target RNA Duplex Required for Archaeal C/D sRNA Guided Nucleotide 2'-O-methylation.

Abstract

Archaeal box C⁄D sRNAs guide the 2'-O-methylation of target nucleotides in both ribosomal and tRNAs. These small non-coding RNAs are characterized by conserved terminal box C⁄D and internal C'⁄D' RNA motifs. Each RNA motif binds three core proteins to establish individual RNP complexes that catalyze the site-specific 2'-O-methylation of target nucleotides. Specificity of nucleotide modification is determined by target RNA base pairing with complementary sRNA D or D' guide sequences. The fifth target nucleotide upstream from the D or D' box within the guide:target RNA is then methylated by the core proteins. In vitro assembly of Methanocaldococcus jannaschii sR8 box C⁄D RNA with recombinant core proteins, L7, Nop56⁄58, and fibrillarin produces a methylation-competent sRNP complex. This model box C⁄D sRNP has now been used to determine the structural features of the guide:target RNA duplex that are important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was essential for nucleotide methylation. Mismatched bases within the guide:target RNA duplex also disrupted target nucleotide methylation. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA helix as target deoxy-oligonucleotides possessing a target ribonucleotide were not methylated. Methylation specificity at the base paired guide:target nucleotide was compromised by elevated Mg2+ concentrations. In high divalent cation concentrations, target nucleotides not hydrogen bonded to the guide nucleotide were nevertheless methylated. Interestingly, D and D' target RNAs were methylated to different levels when deoxynucleotides were inserted within the target RNA or when target methylation was carried out in elevated Mg2+ concentrations. These results suggested that structural features unique to the box C⁄D and C'⁄D' RNPs affect their nucleotide methylation capabilities. Finally, the ability of the sR8 box C⁄D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested an intrinsic ability of this archaeal RNA:protein enzyme to unwind double-stranded target RNAs prior to nucleotide modification.