Search

In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (Psi) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative Psi 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 Psi formation in rRNA. We also identified four single Psi-guide box AGA RNAs. We determined target sites for these putative Psi-guide RNAs and confirmed that the predicted Psi 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.

In a previous investigation of the rDNA region in Tetrahymena pyriformismitochondrial DNA, we identified a putative tRNAMetgene [Heinonen et al. (1987) J. Biol. Chem. 262, 2879–2887]. On the basis of Northern hybridization analyses, we suggested that this gene is expressed, even though the resulting tRNA would be unusually small and have an atypical dihydrouridine stem‐loop domain. We report here the complete nucleotide sequence and post‐transcriptional modification pattern of this tRNAMet, confirming its predicted primary structure and supporting the view that this structurally aberrant species functions in translation in T. pyriformismitochondria.

In Crithidia fasciculata, a trypanosomatid protozoan, the nuclear‐encoded ‘28S’ rRNA is multiply fragmented, comprising two large (cand d) and four small (e, f, gand j) RNA species. We have determined that the coding sequences for these RNAs (and that of the 5.8S rRNA, species i) are separated from one another by spacer sequences ranging in size from 31 to 416 bp. Coding and spacer sequences are presumably co‐transcribed, with excision of the latter during post‐transcriptional processing generating a highly fragmented large subunit (LSU) rRNA. Secondary structure modelling indicates that the C. fasciculataLSU rRNA complex (seven segments, including 5.8S rRNA) is held together in part by long‐range intermolecular base pairing interactions that are characteristic of intramolecularinteractions in the covalently continuous LSU (23S) rRNA of Escherichia coli. At least one functionally critical region (encompassing the α‐sarcin cleavage site) is contained in a small RNA species (f) rather than in one of the two large RNAs. Within a proposed secondary structure model of C. fasciculataLSU rRNA, discontinuities between the different segments (created by spacer excision) map to regions that are highly variable in structure in covalently continuous LSU rRNAs. We suggest that ‘rRNA genes in pieces’ and discontinuous rRNAs may represent an evolutionarily ancient pattern.

In contrast to earlier proposals, recent evidence suggests that trans‐spliceosomes in trypanosomatid protozoa may contain a homolog of U1 small nuclear (sn) RNA (Schnare, M.N. and Gray, M.W. (1999) J. Biol. Chem. 274, 23 691–23 694). However, the candidate trypanosomatid U1 snRNA is unconventional because it lacks the highly conserved stem/loop II present in all other U1 snRNAs. Trypanosomatids also possess a unique spliced leader‐associated (SLA) RNA of unknown function. We present the complete sequence of the SLA RNA from Crithidia fasciculataand propose that it may contribute a U1 snRNA‐like stem/loop II to the trans‐spliceosome.