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

Abstract

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 kinkturn 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 39-terminal ACA sequence, and many contain only a single 39 guide region rather than the canonical 59 and 39 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.

RNA-guided isomerization of uridine to pseudouridine—pseudouridylation

“…from 11 sites in bacterial rRNA (stand-alone enzymes) to ~50 in yeast and ~100 in mammalian rRNA (both RNA-guided) 102,103 . Despite the extensive pseudouridylation of all tRNAs in all species, archaea offer the only known example of RNA-guided modification of tRNA104,105 . In summary, only stable noncoding RNAs are targets of RNA-guided pseudouridylation, which shall be the focus of this review.…”

Complete genome sequence of Pyrobaculum oguniense

“…There are 47 annotated tRNA genes, plus a single tRNA pseudogene. Also included are 83 predicted C/D box sRNA genes and nine additional H/ACA-like sRNA, each of which has been transcriptionally validated [31]. The non-coding RNA content of the P.…”

“…C/D box sRNA genes were identified computationally using Snoscan [30] with extensions supported by transcriptional sequencing [51]. H/ACA-like sRNA genes were identified using transcriptionally-supported homology modeling of experimentally validated sRNA transcripts [31]. CRISPR repeats were identified using CRT [32] or CRISPR-finder [33], with strandedness established by transcriptional sequencing.…”

Ribonucleoproteins in Archaeal Pre-rRNA Processing and Modification

“…Both computational and experimental approaches have been used to detect guide sRNAs in various archaeal species [42, 43, 61–67]. Computational approaches used to identify these sRNAs take advantage of the conserved length and sequence elements and homologies with known snoRNAs or sRNAs in other eukaryotes and Archaea [63, 65, 67]. However, the relatively small sizes of the conserved sequence features of the sRNAs and the low degree of conservation of the sequence outside these features have presented challenges to the identification of sRNAs in Archaea.…”