Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus

Abstract

In a specialized cDNA library from the archaeon Archaeoglobus fulgidus we have identified a total of 86 different expressed RNA sequences potentially encoding previously uncharacterized small non-messenger RNA (snmRNA) species. Ten of these RNAs resemble eukaryotic small nucleolar RNAs, which guide rRNA 2'-O-methylations (C/D-box type) and pseudouridylations (H/ACA-box type). Thereby, we identified four candidates for H/ACA small RNAs in an archaeal species that are predicted to guide a total of six rRNA pseudouridylations. Furthermore, we have verified the presence of the six predicted pseudouridines experimentally. We demonstrate that 22 snmRNAs are transcribed from a family of short tandem repeats conserved in most archaeal genomes and shown previously to be potentially involved in replicon partitioning. In addition, four snmRNAs derived from the rRNA operon of A. fulgidus were identified and shown to be generated by a splicing/processing pathway of pre-rRNAs. The remaining 50 RNAs could not be assigned to a known class of snmRNAs because of the lack of known structure and/or sequence motifs. Regarding their location on the genome, only nine were located in intergenic regions, whereas 33 were complementary to an ORF, five were overlapping an ORF, and three were derived from the sense orientation within an ORF. Our study further supports the importance of snmRNAs in all three domains of life.

Figure 1

The four pseudouridylation guide RNAs: Afu-4, Afu-46, Afu-52, and Afu-190 from A. fulgidus. (A) Potential base-pairing interaction with 16S or 23S rRNA involving each pseudouridylation pocket. The predicted site of pseudouridylation is denoted by an arrow, and its location within the cognate rRNA is indicated by numbering. Its distance from the ACA/AGA- (or H-) box (usually between 14 and 16 nt) is indicated also. The snmRNA sequence in a 5′-to-3′ orientation is shown in the upper strand, with the apical part of the long hairpin domain schematized by a solid line. (B) Verification of predicted pseudouridylation sites in A. fulgidus rRNA by primer extension. Total cellular RNA samples submitted or not to CMC modification (lanes R and N, respectively) were analyzed by primer extension using ³²P-labeled primers complementary to an appropriate segment of 16S or 23S rRNA. Predicted sites are denoted by arrowheads, additional pseudouridines thus far without a known cognate H/ACA guide are denoted by arrows, and cDNA sizes (in nt) are indicated in the margin.

Figure 2

Location (A–C), Northern blot analysis (D), and sequence (E) of repeats from A. fulgidus and other archaeal species. (A–C) Location of repeats on locus 1, 2, and 3 of the A. fulgidus genome (not drawn to scale) in respect to annotated ORFs or the tRNA-Leu-2 gene. Repeats are shown by yellow or green triangles, which indicate the direction of transcription. The relative position of cDNA clones representing snmRNAs is indicated by red arrows, protein or tRNA genes are shown by light gray arrows, and putative promotor sequences for the three repeat loci are indicated by blue boxes. (D) Northern blot analysis of repeated sequences. For each locus, an oligonucleotide derived from the respective repeat motif was used as a probe. (E) Sequence alignment of repeats from three loci in A. fulgidus (Upper) compared with short regularly spaced repeats of four different archaeal species (Lower). Deviations from the A. fulgidus repeat sequence are indicated in red. The total number of all related repeats as well as the number of loci to which repeats are mapped are indicated on the right.