With the establishment of sRNAs in two Sulfolobus species, we next asked how ubiquitous this class of RNAs might be among the Archaea. Fortunately, genome sequence is available from species covering a wide range of phyla, including members from both main divisions of the Archaea, the Crenarchaea (Sulfolobus solfataricus, Aeropyrum pernix), and the Euryarchaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidis, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus). Evidence of methylation guide sRNAs in any of the Euryarchaeal species would imply that this feature originated before the split between Archaea and Eukarya. In searching these genomes for guide sRNAs, I found strong candidates in all but one of these seven species (see sRNA sequence alignments in Figures 5.5, 5.6, 5.7, & 5.8). No strong candidates were found in the genome of M. thermoautotrophicum.
The search of the M. jannaschii genome gave eight strong hits. Strikingly, all eight candidates contain precise canonical box features for the C (ATGATGA), D' (CTGA), C' (TGATGA), and D boxes (CTGA) (Figure 5.5). All contain 5-9 bp long terminal stems, and each has one or two rRNA complementary guide sequences of 9-13 nt. Again, as seen for the Sulfolobus guide RNAs, these RNAs are extremely compact, with very small gaps (1-7 nt) between box features and guide regions. I plan to perform primer extensions with sRNA-specific primers and M. jannaschii total RNA (kindly provided by James Brown) to confirm these predictions. I also plan to assay 4-6 sites of predicted methylation by the dNTP concentration-dependent primer extension assay. However, based on the strong feature conservation observed in the candidates, we believe the M. jannaschii C/D box sRNAs are also involved in guiding methylation of ribosomal RNA.
Based on high overall score and conserved sequence characteristics, I identified with high confidence four sRNA candidates from A. fulgidis (Figure 5.5). Two predictions had 4-5 bp terminal stems. Numerous other candidates that had one or more imperfect features were found, but in the absence of ribose methylation information for A. fulgidis rRNA, their authenticity remains suspect.
Aeropyrum pernix, which was the only Crenarchaea for which I had a complete genomic sequence to search, produced 29 candidate sRNAs (25 of these are shown in Figure 5.6). The A. pernix sRNA candidates show relatively relaxed sequence feature conservation and spacing, more similar to the crenarchaeal Sulfolobus sRNAs identified (Figure 5.4) than the euryarchaeal sRNAs (Figures 5.5, 5.7, & 5.8). This may be a general trend that is better supported as sRNAs from other archaeal species are identified.