RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

Abstract

The bulge-helix-bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularization of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5' external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

“…RNA-seq methodology revealed an abundant genome-wide presence of circRNAs in the transcriptome of the archaeon Sulfolobus solfataricus 10 such as tRNA introns and rRNA processing intermediates, as well as several protein-coding genes, and many smaller non-coding RNAs (e.g., box C/D RNAs). This genome-wide study is also in agreement with earlier studies that have revealed the presence of circular forms in excised tRNA-introns, 11,12 introns of rRNAs, 13,14 rRNAs processing intermediates 15 and box C/D RNAs in archaea. 16 Note that box C/D RNAs guide site-specific modification (2 0 -0-methylation) of rRNA and small nuclear RNA in eukaryotes and tRNA in archaea.…”

“…Formation of the circular 5S rRNA could be compatible with the proposed processing mechanism of the archaeal pre-5S-RNA possibly 31 but it is unclear whether this potential circular form corresponds to an additional processing intermediate or the mature 5S rRNA. 15 When linear isoforms of the naturally occurring C/ D box and 5 S rRNAs were used in binding (Fig. 3A) and circularization ( Fig.…”

“…As 5S rRNA interacted with Pab1020 in cell-free extracts and its circular form has been previously observed, 10 this enzyme may participate in 5S rRNA pre-processing via a circular intermediate, as previously proposed for 16S and 23S rRNAs. 15 Circularization of physiologically significant RNA molecules by an archaeal RNA ligase Pab 1020…”

The heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge–helix–bulge motifs of joined tRNA halves

“…This step is followed by the joining of the tRNA exons catalyzed by a tRNA splicing-specific ligase in Eukarya; however, little is known about the tRNA ligation mechanism in Archaea (3). The archaeal splicing endonuclease recognizes a consensus bulgehelix-bulge (BHB) structure that is found at the intron-exon junctions of intron-containing tRNA (4), rRNA (5,6), and mRNA (7). The BHB motif exhibits pseudo-twofold symmetry with the two 3-nt bulges present on the same minor groove face of the central 4-bp helix, termed the 3-4-3 BHB (8).…”

CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genusSulfolobus

“…45 Northern blot analysis of CRISPR pre-RNA revealed the presence of antisense transcripts for each of the S. acidocaldarius CRISPR loci. 55 Bi-directional transcription was also experimentally verified in S. solfataricus CRISPR loci 26,55,67,68 and was confirmed by analyzing publicly available transcriptome data. 69 However, the loci were primarily transcribed from the leader strand.…”