Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis

Abstract

In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (Ψ) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative Ψ synthase, Cbf5p. In this study we have identified Cbf5p-associated RNAs in Euglena gracilis, an early diverging eukaryote, by immunoprecipitating Cbf5p-containing complexes from cellular extracts. We characterized one box H/ACA-like RNA which, however, does not appear to guide Ψ formation in rRNA. We also identified four single Ψ-guide box AGA RNAs. We determined target sites for these putative Ψ-guide RNAs and confirmed that the predicted Ψ modifications do, in fact, occur at these positions in Euglena rRNA. The Cbf5p-associated snoRNAs appear to be encoded by multicopy genes, some of which are clustered in the genome together with methylation-guide snoRNA genes. These modification-guide snoRNAs and snoRNA genes are the first ones to be reported in euglenid protists, the evolutionary sister group to the kinetoplastid protozoa. Unexpectedly, we also found and have partially characterized a selenocysteine tRNA homolog in the anti-Cbf5p-immunoprecipitated sample.