The identification of C/D box sRNAs from a wide spectrum of Archaeal genera allowed us to search for guide sequences capable of directing methylation to homologous sites within the respective 16S and 23S rRNAs. Based on CLUSTAL [Higgins et al., 1992] alignments of the rRNA sequences, a total of 19 sites of conserved methylation were identified; of these, 14 were shared between two genera and five were shared between three genera. In nearly all of the 19 cases, the sequence similarity between sRNAs that direct methylation to a homologous site is limited to only the guide region that targets the methylation. Moreover, the directing guides can be either both in the same position (i.e., both D' or both D box associated) or in different positions (i.e., one D' and the other D box associated). In only one instance have we detected strong end-to-end sequence similarity between two sRNAs from different Archaeal genera: Pho sR39 and Mja sR06.
Based on these and other data we cannot tell if guides that direct methylation to homologous sites in rRNA are related to each other by common ancestry (i.e., homology) or by sequence convergence. If the relationship is by homology, it implies that guide and target sequences can co-evolve over long periods of evolutionary time if the selection for the methylation is sufficiently important (i.e., for the folding and stabilization of rRNA structure). Alternatively, the rapid accumulation of substitutions in guide sequences (relative to substitutions in rRNA targets) as seen in numerous Pyrococcal families, coupled with weak selection for methylation at a particular position, might suggest that many of the conserved sites for methylation between genera arose through convergence. Once a particular guide sequence lost its target specificity through the accumulation of nucleotide substitutions, it would become free to "explore sequence space" in order to identify more favorable interactions with rRNA or other types of RNA. We have also noted eight examples where sRNAs from different genera appear to direct methylation to nearby but not precisely identical rRNA sites. In these cases, it is possible that base pairing between the sRNA and rRNA during ribosome biogenesis (i.e., chaperone function) may be more important than the precise positioning of a methyl group within the mature rRNA.