FIGURE 5.

Target nucleotides positioned within highly folded target RNAs are methylated. D target RNAs possessing target nucleotides within highly folded RNA secondary structures were incubated with in vitro assembled sR8 box C/D sRNP and the methylation of target nucleotides assessed. (A) D target RNAs folded into hairpin-loop structures with the target nucleotide positioned within the RNA hairpin (stem targets) or RNA loop region (loop targets). Target RNA sequences base-pairing with the sR8 D guide region are boxed and target nucleotides are indicated with stars. Brackets indicate the RNA hairpin region disrupted in the control stem and loop target RNAs. Target RNA melting temperatures (Tm) are indicated at the side. (B) Incorporation of ³H–CH₃ into D target RNAs. Levels of D target nucleotide methylation (various folded hairpin-loop structures indicated by assorted bar coloration) are presented in parentheses as the percent of nonfolded, control D target RNAs (black bars) with methylation incubation temperatures indicated below.