In Crithidia fasciculata, a trypanosomatid protozoan, the nuclear‐encoded ‘28S’ rRNA is multiply fragmented, comprising two large (c and d) and four small (e, f, g and 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. fasciculata LSU rRNA complex (seven segments, including 5.8S rRNA) is held together in part by long‐range intermolecular base pairing interactions that are characteristic of intramolecular interactions 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. fasciculata LSU 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.

We present the cloning and sequence analysis of the nuclear-encoded Crithidia fasciculata small subunit (SSU) rRNA gene, the longest (2,206 bp) such gene yet characterized by direct sequence analysis. Much of the sequence can be folded to fit a phylogenetically conserved secondary structure model, with the additional length of this gene being accommodated within discrete variable domains that are present in eukaryotic SSU rRNAs. On the basis of sequence comparisons, we conclude that Crithidia contains the most highly diverged SSU rRNA described to date among the eukaryotes, and therefore represents one of the earliest branchings within the eukaryotic primary kingdom.

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.