Discovery of Pyrobaculum small RNA families with atypical pseudouridine guide RNA features

In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an essential step in ribosomal RNA maturation. While readily modeled and identified by computational methods in eukaryotic species, these guide RNAs have not been found in most archaeal genomes. Using high-throughput transcriptome sequencing and comparative genomics, we have identified ten novel small RNA families that appear to function as H/ACA pseudouridylation guide sRNAs, yet surprisingly lack several expected canonical features. The new RNA genes are transcribed and highly conserved across at least six species in the archaeal hyperthermophilic genus Pyrobaculum. The sRNAs exhibit a single hairpin structure interrupted by a conserved kink-turn motif, yet only two of ten families contain the complete canonical structure found in all other H/ACA sRNAs. Half of the sRNAs lack the conserved 3'-terminal ACA sequence, and many contain only a single 3' guide region rather than the canonical 5' and 3' bipartite guides. The predicted sRNA structures contain guide sequences that exhibit strong complementarity to ribosomal RNA or transfer RNA. Most of the predicted targets of pseudouridine modification are structurally equivalent to those known in other species. One sRNA appears capable of guiding pseudouridine modification at positions U54 and U55 in most or all Pyrobaculum tRNAs. We experimentally tested seven predicted pseudouridine modifications in ribosomal RNA, and all but one was confirmed. The structural insights provided by this new set of Pyrobaculum sRNAs will augment existing models and may facilitate the identification and characterization of new guide sRNAs in other archaeal species.