Elucidating the role of H/ACA-like RNAs in trans-splicing and rRNA processing via RNA interference silencing of the Trypanosoma brucei CBF5 pseudouridine synthase

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.