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.
Barth
Sarit
S
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Hury
Avraham
A
Liang
Xue-Hai
XH
Michaeli
Shulamit
S
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017353:Gene Deletion; D020868:Gene Silencing; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D010641:Phenotype; D011560:Pseudouridine / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014345:Trypanosoma / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014404:Tubulin / Q000737:chemistry
D008869:Microtubule-Associated Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D011560:Pseudouridine; D012313:RNA; D006836:Hydro-Lyases; C082278:CBF5 protein, S cerevisiae; C022479:pseudouridylate synthetase