The genomes of three closely related Pyrococcal species (P. horikoshii, P. furiosus, and P. abyssi) have been sequenced. This allows powerful comparative analysis of genes that are recognizably homologous because they are recently diverged. However, the evolutionary distance between these particular species is great enough that syntenic DNA without selective pressure is not conserved due to mutational drift. For our purposes, alignments between highly similar sequences that do not code for proteins generally indicate RNA genes, regulatory elements, or other biologically important sequences. We used this information to support our Pyrococcus sRNA predictions without experimentation.
The searches of the P. horikoshii, P. furiosus, and P. abyssi genomes identified 50, 52 and 56 putative C/D box sRNAs, respectively. The complete set of P. horikoshii sequences are presented in Figure 5.7. Most of the D and D' guide sequences in the 50 different P. horikoshii sRNAs exhibit at least one extended complementarity to a known stable RNA. Because of the close relationship between the three species, it was easy to associate the sRNAs by sequence similarity into 57 homologous groups. Alignments of the first ten homolog groups appear in Figure 5.8. Members of the same group ranged from 80 to 98 percent identity in end-to-end sequence alignments. Forty-six groups were found in all three species, 11 were found in only two species, and two were unique to a single species. Of the 50 P. horikoshii sRNAs, only Pho sR33 is unique; the other 49 have at least one homolog in one of the other Pyrococcal species. For each Pyrococcal homolog group, sequence similarity extends over both the D' and D box guides. Except for a few notable instances, the respective D' and D box guides appear to target homologous RNA methylation sites across the three species. In the cases of Pfu sR5 and Pfu sR7, a single base insertion upstream of the D' box appears to have slightly shifted the target methylation site by one nucleotide, relative to the sRNAs for the other two species (Figure 5.8). In three cases, complementary regions have changed by three or more nucleotides relative to the other two homologous sRNAs, likely changing the region or molecule targeted for methylation. Observations of this type support the view that methylation target selection is an ongoing, dynamic process.
Although we have identified regions of sequence complementarity between most of the Pyrococcal sRNA guides and stable RNAs, we cannot be sure which of the matches are functionally significant and which are fortuitous. We suspect that most may be functional since in almost all cases, the guide target complementarities are conserved within the Pyrococcal sRNA families (and in some instances with other genera of archaea).