Three Small Nucleolar RNAs Identified from the Spliced Leader-Associated RNA Locus in Kinetoplastid Protozoans

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.

Section: Sla1 Depletion Eliminates the ⌿28 Of The Sl Rna And Also Affmentioning

confidence: 99%

Section: Modifications On U Snrnas Are Carried Out In Part By H/aca-imentioning

confidence: 99%

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

Barth¹,

Hury²,

Liang

et al. 2005

“…It was found that the trypanosomatid snoRNAs do not obey the general methylation rules. More specifically, the methylation sites corresponding to the duplex formed between the snoRNA and the target site are located either 1 or 6 nucleotides upstream from box D or DЈ (34). More recently, 17 box C/D snoRNAs were analyzed from T. brucei (35) by immunoprecipitation using antibodies against the cloned T. brucei fibrillarin protein.…”

mentioning

confidence: 99%

Expression Studies on Clustered Trypanosomatid Box C/D Small Nucleolar RNAs

Xu¹,

Liu²,

López-Estraño³

et al. 2001

We analyzed three chromosomal loci of the trypanosomatid Leptomonas collosoma encoding box C/D small nucleolar RNAs (snoRNAs). All the snoRNAs that were analyzed here carry two sequences complementary to rRNA target sites and obey the ؉5 rule for guide methylation. Studies on transgenic parasites carrying the snoRNA-2 gene in the episomal expression vector (pXneo) indicated that no promoter activity was found immediately adjacent to this gene. Deleting the flanking sequences of snoRNA-2 affected the expression; in the absence of the 3-flanking (but not 5-flanking) sequence, the expression was almost completely abolished. The snoRNA genes are transcribed as polycistronic RNA. All snoRNAs can be folded into a common stem-loop structure, which may play a role in processing the polycistronic transcript. snoRNA B2, a member of a snoRNA cluster, was expressed when cloned into the episomal vector, suggesting that each gene within a cluster is individually processed. Studies with permeable cells indicated that snoRNA gene transcription was relatively sensitive to ␣-amanitin, thus supporting transcription by RNA polymerase II. We propose that snoRNA gene expression, similar to protein-coding genes in this family, is regulated at the processing level.

“…In addition, the SLA1 locus also carries both SLA1 and three C/D RNAs (27,35). All the clusters analyzed so far are transcribed as polycistronic snoRNA precursors by RNA polymerase II RT-PCR was performed as described under "Experimental Procedures" with oligonucleotides Neo and B2-tag-long.…”

Section: Discussionmentioning

confidence: 99%

Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

Liang

Ochaion

et al. 2004

Trypanosomatid small nucleolar RNA (snoRNA) genes are clustered in the genome. snoRNAs are transcribed polycistronically and processed into mature RNAs. In this study, we characterized four snoRNA clusters in Leptomonas collosoma. All of the clusters analyzed carry both C/D and H/ACA RNAs. The H/ACA RNAs are composed of a single hairpin, a structure typical to trypanosome and archaea guide RNAs. Using deletion and mutational analysis of a tagged C/D snoRNA situated within the snoRNA cluster, we identified 10-nucleotide flanking sequences that are essential for processing snoRNA from its precursor. Chromosome walk was performed on a snoRNA cluster, and a sequence of 700 bp was identified between the first repeat and the upstream open reading frame. Cloning of this sequence in an episome vector enhanced the expression of a tagged snoRNA gene in an orientation-dependent manner. However, continuous transcript spanning of this region was detected in steady-state RNA, suggesting that snoRNA transcription also originates from an upstreamlong polycistronic transcriptional unit. The 700-bp fragment may therefore represent an example of many more elements to be discovered that enhance transcription along the chromosome, especially when transcription from the upstream gene is reduced or when enhanced transcription is needed.In eukaryotes, pre-rRNA transcript undergoes numerous site-specific modifications before cleavage. Two prevalent modifications, 2Ј-O-methylation and conversion of uridine to pseudouridine, are guided by C/D box and H/ACA box small nucleolar RNAs (snoRNAs), 1 respectively (1-3). The C/D snoRNAs carry short conserved motives, as do the C box (RUG-AUGA) and D box (CUGA), near the 5Ј and 3Ј ends, respectively, as well as the internal DЈ and CЈ boxes. Sequences (Ͼ10 nt) upstream from the D and/or DЈ box can interact with the target RNA by perfect base pairing. The 5th nt on the target RNA upstream from the D or DЈ box in the duplex is methylated, which is known as the "ϩ5 rule" (1). The H/ACA snoRNAs that guide pseudouridylation carry two hairpin structures linked by the H box (AnAnnA) in the hinge region and an ACA box 3 nt upstream from the 3Ј end. The internal loop interacts with the target RNA to form the pseudouridylation pocket. The uridine on the target RNA to be isomerized is located usually 14 -16 nts upstream from the ACA and/or H box (2, 3).In vertebrates, all guide snoRNAs are intronic (4, 5). Most yeast snoRNAs are encoded by independent genes, some of which are found in clusters. However, seven intronic genes were also described (6). In plants, most snoRNAs are clustered and are independently transcribed, but intronic snoRNA gene clusters also exist (7-9). The intronic snoRNA genes are transcribed from the promoter of host genes by RNA polymerase II (10, 11). The polycistronic snoRNAs in yeast are transcribed by polymerase II from their own promoters carrying the TATA box and the binding site of Rap1p, a transcription factor involved in ribosomal protein expression (6). In plants, promoters exp...