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.

Figure 1

Ligated RNA species from processing stems of the ribosomal operon of A.fulgidus (A and C–E) and S.solfataricus (B and F–H). (A) Sequence of the A.fulgidus rRNA operon. Black boxes indicate mature rRNAs and thin black lines depict spacer sequences flanking mature rRNAs in pre-16S and pre-23S rRNAs. tRNA^Ala is in blue letters and underlined. Bipartite sequences of the ligated 16S-D RNA and 23S-D RNA species, delineated by analysis of the cDNA clones and northern blot and primer extension assays, are highlighted in red and green, respectively, with the terminal heterogeneity observed for 23S-D cDNA clones denoted by dotted green underlining. (B) Sequence of the S.solfataricus rRNA operon. The sequence of the ligated 16S/23S-D RNA species is highlighted in blue; the other symbols are as in (A). (C–H) Proposed pre-rRNA processing pathway in A.fulgidus (C–E) and S.solfataricus (F–H). Mature rRNAs are represented by thick black lines, precursor sequences by thin lines. BHB motifs are shown within processing stems of rRNAs; the cleavage sites within BHB motifs are indicated by a red arrow. Further processing steps of ligated RNA species are shown by purple arrows. Other symbols are as in (A) and (B).

Figure 2

Northern blot analysis showing expression of 16S-D RNA from A.fulgidus (A) and 16S/23S-D RNA from S.solfataricus (B). (A) Northern blot analysis using an oligonucleotide bridging the proposed fusion site in 16S-D RNA (16S-D) and an oligonucleotide probe specific for tRNA^Ala, located 3′ to the 16S-D RNA (tRNA-Ala). Mature RNAs (16S-D, tRNA^Ala) are indicated by arrows, precursor-RNAs by stars. (B) Northern blot analysis of 16S/23S-D RNA from S.solfataricus. Oligonucleotides bridging the 16S (left) or 23S (right) fusion sites were used as hybridisation probes. The mature 16S/23S-D RNA with a size of 268 nt is indicated by an arrow, the precursor RNA by a star. (Right) Length of RNAs as measured by an RNA length marker.

Figure 3

Structure analysis of 16S-D RNA from A.fulgidus (A and B) and 16S-D/23S-D RNAs from S.solfataricus (C) and S.acidocaldarius (D). (A) Structure prediction of 16S-D RNA by the mfold program and chemical probing data as deduced from (B). Indicated are the C, D′, C′ and D boxes of 16S-D RNA also observed in canonical archaeal C/D box snoRNAs (26,27). The structural hallmark of C/D RNAs, a K-turn motif (29–31) formed by C′/D box apposition, is denoted by a red box. Bases found to be strongly, medium or weakly reactive to chemical probes are shown by red, orange and yellow circles and squares, respectively. Bases whose reactivity could not be determined are indicated by grey circles and squares. The location of the splice site in 16S-D RNA (between positions 139 and 140), including the location of the 16S rRNA, is also shown. (B) Autoradiograms of chemical probing analysis of stems II and III from 16S-D RNA of A.fulgidus. U, C, G, A, sequencing lanes; contr., control lane, no chemical probes added; KE, DMS, CMCT, respective chemical probe added. (Left) The reactive bases in the single-stranded regions in between stems and reactive bases in loop regions of stems are shown. (C) Computer modelling of S.solfataricus 16S/23S-D RNA. The conserved box D motif at the base of stem II is boxed (nucleotide positions corresponding precisely to the box C′ motif present in A.fulgidus are overlined). The red arrowhead denotes the 3′-end of the in vitro synthesised transcript, designated 16S/del23S-D RNA, tested in the Figure 4D gel shift assay. The K-turn at the base of the pre-23S processing stem is delineated by a red box (boxes C′ and D are boxed by black lines). (D) The presumptive L7AE recognition motif (indicated by a red box) at the base of the pre-23S rRNA processing stem of S.acidocaldarius pre-rRNA. Boxes C′ and D are shown by black lines. The previously proposed processing stem (8) extends below the positions denoted by arrows.

Figure 4

Interaction of the ligated RNA species with L7AE protein detected by electrophoretic mobility shift assay and structure of the C/D box motif within the sR6 snoRNA from P.abyssi. In vitro transcribed, ³²P-labelled RNAs were incubated with increasing concentrations of recombinant P.abyssi L7AE protein and the resulting complexes resolved on a native 8% polyacrylamide gel. Left lanes, no protein. (A) (Left) Structure of the C/D box motif located within sR6 snoRNA from P.abyssi; (right) control mobility shift assay with P.abyssi sR6 snoRNA. In lanes 1–9, the sR6 snoRNA was incubated with L7AE protein at concentrations of 0.05, 0.1, 0.25, 0.50, 0.75, 1, 1.25, 1.5 and 2 µM, respectively. (B) Interaction of A.fulgidus 16S-D RNA with the L7AE protein. (C) Interaction of S.solfataricus 16S/23S-D RNA with the L7AE protein. (D) Interaction of the 16S-D domain (as delineated in Fig. 3C, red arrow) of S.solfataricus designated 16S/del23S-D RNA with the L7AE protein. (B–D) In lanes 1–10, incubations were performed with L7AE protein concentrations of 0.21, 0.28, 0.42, 0.56, 0.84, 1.125, 1.7, 2.25, 3.375 and 4.5 µM, respectively.