Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

Abstract

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...

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

“…Indeed, we have recently demonstrated that the numbers of different snoRNA clusters vary from 1 to 10, and therefore the copy numbers of individual H/ACA genes differ considerably (10). In addition, we noticed a difference in the level of snoRNAs emerging from the same polycistronic RNA, most probably as a result of differential processing efficacy (43). The reduction in the level of H/ACA due to the CBF5 silencing is specific, since no effect on the most abundant C/D snoRNA, the U3, and another C/D (TB11Cs2C2-92 nt snoRNA) was observed (Fig.…”

Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis

“…To date, all of the reported ⌿-guide RNAs from trypanosomatid protozoa (nine from Leptomonas collosoma, three from Trypanosoma brucei, one each from Leishmania seymouri, Leishmania tarentolae, Trypanosoma cruzi, and Crithidia fasciculata; Liang et al 2001;Uliel et al 2004;Liang et al 2004) are also single ⌿-guide RNAs. Based on the E. gracilis ␣Cbf5p immunoprecipitation profile (1 h lane, Fig.…”

RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs

“…The trypanosome snoRNA genes exist in clusters, and as shown in T. brucei (12) and L. major (13), most clusters carry interspersed H/ACA and C/D snoRNAs sequences. snoRNA genes are transcribed polycistronically (14) by RNA polymerase II, and individual RNAs are then processed from the primary transcript by an unknown RNase III enzyme, using information residing in the intergenic regions. Recently, it was demonstrated that the primary snoRNAs transcripts are trans -spliced and polyadenylated (8).…”