Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei - Sequence conservation and implications for 2 '-O-ribose methylation of rRNA

We report the identification of 17 box C/D fibrillarin-associated small nuc leolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei. To sy stematically isolate and characterize these snoRNAs, the T. brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was domed and polyclonal a ntibodies to the recombinant fibrillarin protein were generated in rabbits. Immunoprecipitations from T. brucei extracts with the anti-fibrillarin ant ibodies indicated that this trypanosomatid has at least 30 fibrillarin-asso ciated snoRNAs. We have sequenced seventeen of them and designated them TBR for T. brucei RNA 1-17. All of them bear conserved box C, D, C', and D' el ements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes. Fourtee n of them are novel T. brucei snoRNAs. Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylati on. Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences. Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and ver tebrate methylation guide snoRNAs. Our results indicate that T. brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O-ribose methy lation of rRNA and that the mechanism for targeting the nucleotide to be me thylated at the fifth nucleotide upstream of box D or D' originated in earl y eukaryotes.