Ribonucleoprotein particles guide 2’-O-ribose methylation of rRNA in the Archaeal genus Sulfolobus

In Eukaryotes, specific methylation of 2'-0 ribose moieties in ribosomal RNA (rRNA) is guided by the C/D box family of small nucleolar RNAs (snoRNAs). These RNAs function in association with several nucleolar proteins including fibrillarin (the putative 2'-0 ribose methylase), Nop56, Nop58 and the U4/U6-U5 tri-snRNP protein 15.5kDa/Snul3p. Although significant progress has been made in identifying the protein components of the C/D box snoRNPs, the precise role of each of these proteins in the assembly and methylation function of the complex is poorly understood. In this study, homologs of snoRNA genes were initially identified in both branches of the Archaea. Eighteen small sno-like RNAs (sRNAs) were cloned from the archaeon Sulfolobus acidocaldarius by co-immunoprecipitation with aFD3 and aNOP56, the archaeal homologs of eukaryotic snoRNA-associated proteins. A probabilistic model was trained on these sRNAs to search for more sRNAs in archaeal genomic sequences. Over 200 additional sRNAs were identified in seven archaeal genomes representing both the Crenarchaeota and the Euryarchaeota. The genes encoding the Sulfolobus solfataricus homologues of eukaryotic proteins that are known to be present in C/D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned and the proteins (aFIB, aNOP56 and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of a Sulfolobus methylation guide sRNA were reconstituted in vitro, into a RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56 and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent sitespecific 2'-0-ribose methylation to a short fragment of ribosomal. RNA (rRNA). The presence of aFIB was essential for methylation as suggested by the finding that variant proteins having site-specific amino acid replacements in the putative SAM binding motif of aFIB were able to assemble into an RNP complex but the resulting complexes were defective in methylation activity. These results define the minimal number of components and the conditions required to achieve in vitro, RNA guide directed 2'-0-ribose methylation of a RNA target.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat