To isolate biochemically sno-like RNAs from the archaeon Sulfolobus acidocaldarius, members of the Dennis lab5.4 first cloned the archaeal homologs to the eukaryotic fibrillarin (aFIB) and NOP56 (aNOP) proteins using sequence information from a related species, Sulfolobus solfataricus5.5. The cloned genes were expressed in E. coli and the recombinant proteins were purified and used to raise polyclonal antibodies in rabbits. The two antibody preparations were each highly specific and recognize single polypeptides of the predicted size in total S. acidocaldarius cell extracts (data not shown). Immunoprecipitates formed with crude cell lysate contained a large amount of non-specific RNA. To eliminate this contamination, an ammonium sulfate-glycerol gradient fractionation procedure was introduced. Following the sedimentation step, the antibodies were used first to monitor the size distribution of aFIB and aNOP56 in the gradient fractions by Western blotting [Chamberlain et al., 1998] (Figure 5.1a). Both aFIB and aNOP56 sedimented as a large heterogeneous complex ranging from about 4S to greater than 50S in size; the larger complexes appeared to be enriched for aNOP56 relative to aFIB.
To detect RNAs that associate with aFIB- and aNOP-containing complexes, aliquots from gradient fractions were immunoprecipitated with either anti-aFIB or anti-aNOP56 antibodies. Following immunoprecipitation, total RNA was extracted with phenol from the supernatants and the pellets, and a portion from each was end-labeled with pCp and displayed by denaturing PAGE (Figures 5.1b and c). The most abundant RNAs that were co-immunoprecipitated appear as a family of discrete bands ranging in length from about 50-70 nt. This size class of RNAs, which is substantially shorter than eukaryotic C/D box snoRNAs, was invisible when total cellular RNA was labeled with pCp. To obtain cDNA clones, the RNA precipitated from fraction 5 with anti-aFIB and from fractions 6-8 and 10-13 with anti-aNOP56 were gel purified, ligated to the oligonucleotide oAO30, and used as template for RT-PCR5.6 [Wu et al., 1998]. The PCR products were cloned between the PstI and XhoI sites of pSP72 plasmid.
A total of about 50 clones from each of the two immunoprecipitated RNA pools were sequenced by the Dennis lab. About half had inserts containing random fragments of 16S and 23S rRNA sequence; the other half gave one or more representatives of 18 different sequences which exhibited features characteristic of eukaryotic C/D box snoRNAs (Table 5.4) [Balakin et al., 1996,Kiss-Laszlo et al., 1996,Kiss-Laszlo et al., 1998]. Three of the clones, Sac sR5, sR14, and sR18, were independently recovered from the two separate immunoprecipitations. This was expected since anti-aFIB coprecipitates aNOP and anti-aNOP coprecipitates aFIB from crude cell extracts (data not shown). All 18 clones contained well-defined and highly conserved C(AUGAUGA) and D(CUGA) box motifs located respectively near their 5' and 3' ends. Moreover, all contained recognizable internal C' and D' box motifs, giving the RNAs a dyad repeat structure characteristic of eukaryotic methylation guide snoRNAs [Kiss-Laszlo et al., 1998].
Both the Dennis lab and I used primer extension analysis to confirm the presence of the sRNAs in total RNA extracted from S. acidocaldarius (Figures 5.2a and b; gels pictured are from Dennis lab). I designed the S. acidocaldarius sRNA primers to overlap the common D box motif and extend through the unique guide region and into the C' box motif of sR1 to sR17. I obtained extension products for 15 of 17 sRNA-specific primers. The lengths of the products were within one or two nucleotides of the cloned 5' ends for all but two sRNAs; sR6 and sR8's products were both 4 nt longer than the cloned ends. In separate experiments in the Dennis lab, Southern hybridizations have confirmed the existence of sR1, sR2, sR5, and sR13 encoding single-copy sequences within S. acidocaldarius genomic DNA; the four encoding sequences are not closely linked (data not shown). These data demonstrate that Sulfolobus acidocaldarius contains snoRNA-like C/D box sRNAs.