Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, ∼40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique positions outside the highly conserved domains. Although most of the guide RNAs were found in reiterated clusters, a few single-copy genes were identified. The large repertoire of modifications and guide RNAs in trypanosomes suggests that these modifications possibly play a central role in these parasites.

Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNAprocessing and modification defects that caused lethality. Systematic mapping of 2-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.

Most pseudouridinylation in eukaryotic rRNA and small nuclear RNAs is guided by H/ACA small nucleolar RNAs. In this study, the Trypanosoma brucei pseudouridine synthase, Cbf5p, a snoRNP protein, was identified and silenced by RNAi. Depletion of this protein destabilized all small nucleolar RNAs of the H/ACA-like family. Following silencing, defects in rRNA processing, such as accumulation of precursors and inhibition of cleavages to generate the mature rRNA, were observed. snR30, an H/ACA RNA involved in rRNA maturation, was identified based on prototypical conserved domains characteristic of this RNA in other eukaryotes. The silencing of CBF5 also eliminated the spliced leader-associated (SLA1) RNA that directs pseudouridylation on the spliced leader RNA (SL RNA), which is the substrate for the trans-splicing reaction. Surprisingly, the depletion of Cbf5p not only eliminated the pseudouridine on the SL RNA but also abolished capping at the fourth cap-4 nucleotide. As a result of defects in the SL RNA and decreased modification on the U small nuclear RNA, trans-splicing was inhibited at the first step of the reaction, providing evidence for the essential role of H/ACA RNAs and the modifications they guide on trans-splicing.

Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs.In eukaryotes (2, 9, 17, 42) as well as in archaea (10, 29), the two major rRNA modifications, 2Ј-O-methylation (Nm) and pseudouridylation, are guided by small RNAs, which in eukaryotes are small nucleolar RNAs (snoRNAs). The 2Ј-Omethylations are guided by C/D box snoRNAs, which are named after short motifs known as the C box (RUGAUGA [R designates purines]) and the D box (CUGA). These boxes, together with the short sequences near the 5Ј and 3Ј ends of the RNA, which have the potential to form a K-turn structure, are essential for processing, localization, and stabilization of these molecules (7,19,40,44). The K-turn motif is the binding site for the 15.5-kDa/Snu13 snoRNP protein (26). Such motifs exist in C/D snoRNAs, which guide modification, and also in U3 snoRNP (26) and U4 snRNA (28). Most of the guide RNAs carry internal boxes related to the C and D boxes, known as CЈ and DЈ boxes. The recognition of the target by the guide RNA is based on complementarity of 10 to 21 nucleotides (nt) between these two molecules, located upstream of the D and DЈ sequences. The methylation site is situated 5 nt upstream from the D and DЈ boxes, within the domain of interaction between the snoRNA and the substrate (9, 18).

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position ؊12 (relative to the 5 splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.H/ACA RNA is a group of small nucleolar RNAs that direct pseudouridylation on rRNA and snRNAs. In almost all eukaryotes, these RNAs consist of two stem-loop structures connected by a single-stranded hinge and a tail region carrying the conserved H (AnAnnA) and ACA boxes. Two short motifs of the snoRNA base pair with the target site flanking the uridine to be isomerized (9, 25). In trypanosomes, these guide RNAs have a unique structure, composed of a single stem-loop, and carry an AGA instead of an ACA box (15, 16). All of the H/ACA molecules identified so far in trypanosomes can guide rRNA pseudouridylation; however, molecules that guide analogous modifications on U snRNAs (U1 to U5) have not been identified to date. In higher eukaryotes, the guide RNAs that direct such modifications are localized in special subnuclear domains, the Cajal bodies (6). In trypanosomes, all mRNAs are processed by trans splicing. During the trans-splicing reaction, a common spliced-leader (SL) sequence of 39 nucleotides (nt) is added to the pre-mRNA from a small RNA, the SL RNA. A novel snoRNA, termed SLA1 (SL-associated RNA), in Trypanosoma brucei was initially discovered by virtue of its efficient cross-linking with the bifunctional reagent aminomethyltrimethyl (AMT) to the SL RNA (39). It was originally proposed that SLA1 is the trypanosome U5 sn...