Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

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 upstream-long 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.

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)(8)(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 expressing polycistronic snoRNA clusters have not yet been identified.
Trypanosomatids are unicellular protozoan parasites that diverged very early from their eukaryotic lineage. These organisms possess unique RNA processing mechanisms such as pre-mRNA trans-splicing and RNA editing (16,17). A very unusual characteristic of gene expression in these organisms is the presence of polycistronic transcription. Long transcripts generated by RNA polymerase II are processed by both transsplicing and polyadenylation (18,19). In Leishmania, transcription along the chromosome is bi-directional. A unidirectional transcript can cover as much as one-third or even half of an entire chromosome (20). So far, there is no evidence of the existence of a polymerase II promoter that transcribes proteincoding genes in any of the trypanosomatid species. The only promoters identified so far are polymerase I promoters, polymerase III promoters that transcribe the U small nuclear RNAs and 7SL RNA genes, and the polymerase II promoter that transcribes the spliced leader RNA (SL RNA) gene (21). Two protein-coding genes, the Trypanosoma brucei variant surface glycoprotein and the EP (procyclin), are transcribed by RNA polymerase I (22). In the absence of transcriptional regulation for most of the protein-coding genes, the expression of mRNAs in trypanosomatids is regulated post-transcriptionally by mRNA processing, stability, and translatability (23).
The probable presence of gene clusters carrying both C/D and H/ACA-like RNAs has been reported in studying snoRNA genes in trypanosomatids (24). The first C/D snoRNA identified in Leptomonas collosoma was termed snoRNA-2 and was shown to obey the ϩ5 methylation guiding rule (25). Additional C/D snoRNAs were identified in T. brucei by immunoprecipitation with anti-fibrillarin antibody (26). C/D snoRNAs were also found in a locus encoding for the spliced leader-associated RNA (SLA1 RNA) gene (26,27). Transcription analysis in L. collosoma and T. brucei suggests that snoRNAs are transcribed as polycistronic RNAs by polymerase II (28,29). The snoRNA genes are organized as reiterated gene clusters that are repeated more than five times in the genome (25). No promoter activity upstream from the snoRNA-2 was detected, because expression of the tagged snoRNA-2 gene was dependent on the transcription from the episomal neo gene (28). Although the trypanosome C/D snoRNAs exactly fit the prototype C/D snoR-NAs in eukaryotes and archaea, the trypanosome H/ACA RNAs possess unique features. The first identified trypanosomatid H/ACA RNA, h1 RNA, is composed of a single hairpin, whereas in other eukaryotes, H/ACA RNAs carry two hairpins connected by the H hinge region (24). h1 has an AGA box but not an ACA box at the 3Ј end. Recently, SLA1 was identified as a guide RNA that directs pseudouridylation at position Ϫ12 (relative to the 5Ј splice site) on the SL RNA. Like h1, SLA1 also carries a single hairpin and possesses an AGA box at the 3Ј end (30).
We have demonstrated recently that snoRNA can be silenced in trypanosomatids in L. collosoma, Leishmania major, and T. brucei. The silencing in T. brucei was achieved by conventional RNA i using the T7 opposing system. However, in Leishmania and Leptomonas, the silencing was elicited by antisense RNA. We propose that silencing in L. major and L. collosoma most probably operates by cleaving the mature RNA after its pairing with the antisense transcript by RNase III homologue. However, we detected siRNAs specific to the cleaved transcript, and the amount of antisense RNA needed for silencing was very high, suggesting that siRNAs are probably not catalytic and are not used for cleaving the target RNA via an RNAinduced silencing complex. Although the mechanism of snoRNA silencing is currently unknown, silencing of snoRNAs turned out to be efficient for knocking down C/D but less for inactivating H/ACA RNAs. Silencing of snoRNA-2 almost completely eliminated the modification guided by this RNA (31).
In this study, we analyzed the genome organization of four gene clusters encoding for both C/D and H/ACA RNAs in L. collosoma. Eight novel H/ACA RNAs were identified, and with no exception, all obey the trypanosomatid prototype pseudouridine guidelines, being a single hairpin RNA and carrying an AGA box at the 3Ј end. To determine the sequences essential for the expression of a clustered snoRNA gene, we tagged the B2 gene and performed deletion and mutation analysis by changing the sequences flanking the gene. Mutation of the first 10-nt flanking sequences was deleterious to expression. Moreover, expression of the tagged gene was dependent on its orientation in the vector. In fact, very poor expression of the tagged B2 gene was observed when it was present in the opposite orientation with respect to the neo gene in the vector, because of silencing of the transcript by the antisense effect. Chromosome walk was performed to identify potential regulatory sequences that initiate transcription of the repeated clusters. A 700-bp fragment situated upstream from the first snoRNA repeat and downstream from an open reading frame was shown to enhance transcription in an orientationdependent manner. However, a long transcript from the ORF to the snoRNA cluster was observed, suggesting that snoRNA genes are part of a long polycistronic transcript. This upstream sequence may therefore represent sequences in the genome that can locally promote transcription and hence enhance transcription of genes with special needs.
Plasmid Construction-B2 constructs were generated as described previously (28). Briefly, the B2 snoRNA gene was amplified by PCR with the sense primer 26553, specific to the upstream flanking sequence, and antisense oligonucleotides 35178, 35758, and 35759, to generate constructs containing 15-, 35-, and 75-bp 3Ј-flanking sequences, respectively. The PCR fragments were inserted into the BamHI site of expression vector pX-neo, in two orientations with respect to the neo gene, as depicted in Fig. 3A.
The snoRNA-2 constructs were generated as described previously (28) and are depicted in Fig. 5. 58-1 carries a tagged snoRNA-2 gene flanked by 260-nt upstream and 104-nt downstream sequences and is inserted into the BamHI site of the pX-neo expression vector in the same orientation as the neo transcript. 58-2 possesses the same construct as 58-1, but it is present in the opposite orientation with respect to the neo gene. 2-3 carries the 700-bp sequence found upstream to the first snoRNA repeat and is inserted into the XbaI site of the 58-2 construct, downstream from the snoRNA-2 gene with the same orientation as snoRNA-2, but with an orientation opposite that of the neo gene. 2-4 has the same construct as 2-3, except that the orientation of the upstream sequence is opposite that of the snoRNA-2.
Site-directed Mutagenesis-Site-directed mutations were generated in a two-step process by using PCR. The fragments were amplified using sense primer 26553 and antisense primers 44182 and 44185, carrying 5Ј and 3Ј mutations, respectively. The first-step PCR products were used as a megaprimer to amplify the full-length gene by secondstep PCR with antisense primer 26554. The PCR product was then cloned into the BamHI site of the pX-neo vector. The presence of the mutation was confirmed by sequencing, and the orientation was determined. The constructs carrying the 5Ј and 3Ј mutations were termed M1 and M2, respectively.
Genomic Library Screening-RNPs enriched in the flow-through fraction of a DEAE column served as a source of snoRNAs, as was described previously (25). RNA was separated on a 10% polyacrylamide gel. RNA ranging in size from 70 to 90 nts was excised from a preparative gel and was labeled at the 5Ј end with [␥-32 P]ATP, as described previously (32). Subsequently, the RNA probe was used to screen the -phage genomic library (33). Clean DNA was digested with Sau3A and subcloned into the BamHI site of pBluescript KSϩ. Two clones obtained from two different phages, termed B6 and B7, were sequenced.
To clone the sequence present upstream from the first B2 snoRNA repeats, the L. collosoma genomic library was screened with g2ClaI plasmid (24), which contains the entire repeat of the B2 snoRNA cluster. DNA from positive plague was digested with SalI and SacI and subjected to Southern analysis with a B2-specific probe. A 5.5-kb fragment carrying the B2 cluster was subcloned into the pBluescript KSϩ vector. By using direct sequencing from the vector, we identified the sequence upstream from the first snoRNA cluster. The plasmid that carries this fragment was termed UPSS.
Cell Growth and Establishing Stable Cell Lines-L. collosoma cells were grown as described previously (34). The pX-neo derived constructs were transfected into L. collosoma wild-type cells, as described previously (34). Stable cell lines were selected by G418 and were obtained after 2 weeks.
RNA Preparation and Primer Extension-Total RNA was prepared from the cell lines with TRI Reagent (Sigma). For the primer extension reaction, 10 g of total RNA was annealed with 50,000 cpm end-labeled primer(s) at 55Ϫ60°C for 15 min. After chilling on ice for 2 min, 1 unit of reverse transcriptase (Expand RT, Roche Applied Science) and 1 unit of RNase inhibitor (Promega) were added, and the samples were incubated at 42°C for 90 min. After ethanol precipitation, the samples were analyzed on 6% polyacrylamide, 7 M urea gel.
Northern Analysis-Twenty g of total RNA was separated on 10% polyacrylamide denaturing gel next to the labeled marker (pBR322 MspI digest) and transferred onto a nylon membrane (Hybond). The membrane was hybridized at 42°C for overnight with end-labeled antisense oligonucleotides. After hybridization, the membrane was washed at 42°C twice for 20 min with 2ϫ SSC containing 0.1% SDS.
RT-PCR-The total RNA was extensively treated with DNase inactivation reagent-DNA free (Ambion) to remove the DNA contamination. Reverse transcription was performed on the RNA with antisense primers. The samples were heated for 5 min at 95°C, followed by annealing for 15 min at 60°C. After chilling on ice for 2 min, 1 unit of reverse transcriptase (Expand RT, Roche Applied Science) and 1 unit of RNase inhibitor (Promega) were added, and the reaction was performed at 42°C for 60 min. Next, the cDNA was used in PCR amplification. As a positive control, the PCR was carried out using plasmid as a template, and the negative control consisted of RNA that was directly used for PCR.

Identification of Two Clusters Encoding for Both C/D and H/ACA
Box snoRNAs-To screen the genomic library for snoR-NAs, 70 -90-nt RNAs obtained from the flow-through fraction of a DEAE-Sephacel column (32) were separated from the total RNA and recovered. Next, the eluted RNA was dephosphorylated and labeled with [␥-32 P]ATP. The RNA probe was used to screen an L. collosoma genomic library, and several positive clones were selected for further analysis. DNA was isolated from positive clones, and the DNA was digested by Sua3A1 and subjected to Southern analysis with the RNA probe described above. The hybridizing fragments were isolated from a preparative gel, subcloned, and sequenced. Two novel loci were identified and termed B6 and B7. The structure of the four loci carrying snoRNA genes is depicted in Fig. 1A. Interestingly, all clusters carry both C/D and H/ACA RNAs. An extensive search revealed six novel H/ACA RNAs in previously reported B2 and snoRNA-2 clusters (24,25,28). These new H/ACA RNAs are termed h2 to h9, respectively.
The sequences of the putative H/ACA RNAs were folded using the MFOLD program, www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi, and the predicted secondary structure of the eight new H/ACA RNAs is depicted in Fig.  1B. Interestingly, all of them obey a canonical structure consisting of a single hairpin and carrying an AGA box at the 3Ј end. The sizes of these H/ACA RNAs are around 70 nts. Fig. 2A illustrates the putative interaction domain of four of the eight H/ACA RNAs. The predicted pseudouridines are positioned 15-17 nt from the AGA box, and the lengths of the flanking duplexes formed between the snoRNA and rRNA are 4 -6 bp, suggesting that these RNAs obey the canonical guiding rules for directing pseudouridylation (2,3). To verify the presence of such RNAs, we subjected total RNA to both primer extension and Northern analysis, and the results, presented in Fig. 2, B and C, indicate the existence of these small RNAs. These results, together with the previously identified H/ACA RNA, such as h1 and SLA1, suggest that all H/ACA identified so far in trypanosomatids possess a single hairpin structure and carry a 3Ј AGA box.
Sequences Flanking the snoRNA Genes Governs Its Processing from a Polycistronic Transcript-We reported previously the analysis of a sequence essential for the expression of the snoRNA-2 gene, which is the first snoRNA within its cluster (Fig. 1A). The data suggested that both the 5Ј and 3Ј sequences are essential for proper expression. Deletion of the entire 5Ј flank still afforded expression of the tagged gene yet at a lower level, suggesting that a promoter of the cluster does not lie immediately adjacent to the first gene within a single cluster and may in fact exist upstream from the entire gene clusters (see below). Deletion of the 3Ј flanks affected the expression, and a construct carrying only 82 nt of the upstream and 15 nt of the downstream sequence was expressed but at lower levels (28). In this study, we examined the expression of a snoRNA gene embedded within a cluster. Systematic deletion was performed at the 3Ј end of the gene. In addition, site-directed mutagenesis was performed to change the sequence adjacent to the gene from either side. Constructs carrying the same upstream sequence, covering the entire upstream intergenic region of 82 nt and the varying 3Ј sequence (75, 35 and 15 bp) were generated and cloned into the pX neo plasmid in two orientations with respect to the neo transcript, as illustrated in Fig. 3A. Stable cell lines were established, and RNA from wild-type and cell lines carrying tagged B2 and different mutants was subjected to primer extension analysis using an antisense primer, which can extend both the wild-type and the tagged B2 snoRNAs. The results, presented in Fig. 3A, indicate that the 15-bp 3Ј-flanking sequence is sufficient to efficiently express the tagged B2 gene. Expression of the tagged gene is, however, strictly dependent on its orientation with respect to the neo transcript (Fig. 3A, panel b, lanes 1-3 and 4 -6). We have reported recently (31) that the expression of antisense RNA to the snoRNA gene elicits the degradation of the RNA in a mechanism related to RNA i . This mechanism also explains the poor expression of the tagged B2 gene in cell lines expressing the antisense B2 in the transcriptional direction of the neo gene (Fig. 3A).
To elucidate the flanking sequences that are essential for snoRNA processing, we introduced site-directed mutations to change the first 10 bp upstream and downstream from B2. The results, presented in Fig. 3B, indicate that tagged genes lacking either the first 10 bp upstream or downstream flanking sequences were not expressed, suggesting that these sequences are essential for the expression. To examine whether these mutations affected the processing of the snoRNA but not its transcription, we performed RT-PCR to detect continuous transcripts originating from the episomes that carry the tagged snoRNA transcripts. A sense primer specific for the vector sequence, situated upstream from the tagged B2 gene, and an antisense primer complementary to the tagged B2 coding region were used to amplify cDNA generated using the antisense oligonucleotide. The results (Fig. 3C) indicate that the tagged B2 gene is indeed transcribed into a polycistronic continuous molecule, because the 300-bp fragment was detected in the cell lines carrying the mutations in the flanking sequences (lanes 1  and 2), as well as in the cell line carrying the tagged B2 (lane 3, positive control) but was absent from wild-type cells (lane 4, negative control). The identity of the second molecular weight PCR product is currently unknown. As a control we used the DNA-removed RNA template in a PCR (Fig. 3C, lanes 5-8), and no contamination was detected. These data suggest that the failure to express the tagged snoRNA mutants in the flanking regions stem from the inability to process the polycistronic snoRNA such that it can interact with the binding proteins to generate a stable snoRNP.
Identification of a Sequence Upstream from all B2 Repeats-Our previous study demonstrated that no promoter activity lies immediately upstream from individual snoRNA genes within the cluster. Because the snoRNA genes are reiterated in the genome (24, 28) and carry 5-6 repeats, a promoter may exist upstream from the first repeat. To identify such a regulatory element, we first needed to identify the sequence present upstream from the first cluster. To this end, an L. collosoma genomic library was screened with a B2-specific probe, and DNA isolated from the pure clones was subjected to Southern analysis. We detected a clone, which upon digestion with the restriction enzymes SalI and SacI, showed an additional hybridization fragment (Fig. 4B). This clone can carry either the 5Ј or the 3Ј flank of the gene cluster. We therefore subcloned this fragment and sequenced it. The gene organization is depicted in Fig. 4A. The sequence analysis is presented in Fig. 4C and indicates that this fragment carries the snoRNA cluster as well as the upstream sequence. Bioinformatic analysis identified a part of an ORF with an unknown function as well as a sequence of 700 bp that is situated downstream from the ORF and upstream from the first repeat. This fragment could potentially carry the promoter of the gene clusters. Interestingly, the 700-bp fragment encodes for two perfect units of Ts analogous to polypyrimidine tracts essential for cis-and trans-splicing.
Functional Analysis of the 700 Region as a Potential Promoter of the snoRNA Cluster-The expression of snoRNA in the pX-neo system is orientation-dependent, as we reported previously (28) and also demonstrated in this study (Fig. 3A). We thought to use this observation and enhance the expression of the tagged snoRNA-2 gene by cloning the upstream fragment to the poorly expressed construct. The constructs that were generated are depicted in Fig. 5A. Construct 58-1 carried the tagged snoRNA-2 gene in a transcriptional direction of the neo gene and served as a positive control for maximum expression. Indeed, primer expression indicates strong expression of both the wild-type and the tagged snoRNA-2 gene (Fig. 5B, lane 1). Construct 58-2 carried the snoRNA-2 gene in an opposite orientation with respect to the neo gene, and in this cell line no tagged snoRNA-2 transcript was observed, and the level of the wild-type transcript was also almost completely eliminated (Fig. 5B, lane 2). In addition, the level of h2, which is present in the same construct, was also reduced as a result of silencing (Fig. 5C, lane 2), as we reported previously (31). Interestingly, cloning the 700-bp upstream fragment to the 58-2 construct (construct 2-3) diminished the silencing effect, and the levels of expression of snoRNA-2 and h2 were partially elevated (lane 3 in Fig. 5, B and C). This phenomenon should stem from the increased level of the sense snoRNA-2 transcript generated in this cell line due to the presence of the upstream element. The additional sense snoRNA-2 transcript increases the cellular amount of snoRNA-2, and consequently there was not a sufficient amount of antisense RNA left to pair with all the snoRNA-2 cellular transcripts. If this is indeed the case, we should be able to detect lower levels of antisense snoRNA-2. In addition, because the snoRNA-2 sense transcript originated from the episome may also carry the neo antisense information, silencing of neo transcript should be observed. To this end, cell lines carrying constructs 58-2 and 2-3 were generated and selected on elevated levels of G418. RNA was prepared from the different cell lines and subjected to primer extension, and the results, presented in Fig. 6A, demonstrate that in these cell lines the level of the neo transcript was indeed very different. As expected, the level of the neo transcript increased in cell line 58-2 due to the increase in the copy number of the plasmid. However, in the 2-3 cell line, the level of the neo mRNA first increased, whereas at a very high drug concentrations, the level of neo mRNA decreased, most probably because of the increased levels of antisense RNA to neo that were promoted by the presence of the 700-bp fragment. This phenomenon is clearly observed when comparing the level of neo transcript in cell lines selected under 600 g/ml G418 (Fig. 6A), demonstrating the reduction in the level of neo mRNA in the 2-3 cell line compared with its level in the 58-2 cell line. In all these experiments the level of the h1 RNA was used to control the level of the RNA in the samples, and the RNA used in B is the same as in A. These results strongly suggest that the 700-bp upstream fragment has the ability to promote the expression of downstream genes. Interestingly, the 700-bp element elicited its effect on snoRNA-2 silencing in an orientation-dependent manner, because cloning of this fragment in the opposite orientation with respect to snoRNA-2 (construct 2-4) did not affect silencing (lane 4 in Fig. 5, B and C).

The snoRNA Repeats Are Transcribed into a Polycistronic
Precursor with Upstream ORF-It is now widely accepted that most of the trypanosomatid genes are transcribed as part of a long polycistronic unit. If the 700-bp region functions in vivo as a promoter, it is expected that the region adjacent to this element will be transcriptionally silent. We therefore examined the steady-state level of transcripts around the 700-bp element. RT-PCR was performed with primers specific to different regions, as depicted in Fig. 7A. The results (Fig. 7, B, lane 3, and  C, lane 2) demonstrate the presence of a 700-bp transcript covering the region between the open reading frame and the snoRNA. In addition, we detected RT-PCR products that reflect a continuous transcript (300 nt) from the ORF to the intergenic 700-bp region and from the snoRNA to the intergenic region (340 nt), as demonstrated in Fig. 7C, lanes 1 and 2. Note the absence of DNA contamination (Fig. 7, B, lane 1, and C, lane 3). These results suggest that the 700-bp region is actively transcribed.

DISCUSSION
In this study we enlarged the repertoire of H/ACA snoRNAs described so far in trypanosomatids and provided evidence of the role of snoRNA flanking regions for processing the long pre-snoRNA precursors. Moreover, we described the identification of a 700-bp fragment situated between the ORF and the first snoRNA cluster within the snoRNA repeats, and we demonstrated the ability of this fragment to enhance the expression of a tagged snoRNA gene in an orientation-dependent manner. However, this element cannot be considered as a classical promoter because a continuous transcript covering this region was detected that spans this region.
All trypanosomatid snoRNA genes identified so far are present in clusters in the genome, which encode for both C/D and H/ACA RNAs, as we described previously (24). 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 The extension products were separated on 6% polyacrylamide denaturing gel. The identity of the products is marked with arrows and indicated. The arrows above the lanes indicate the orientation of the tagged gene with respect to the neo gene in the vector. Lanes 1-6, total RNA from the cell lines carrying constructs 1-6, respectively, as illustrated in panel a. B, mutational analysis of the sequences flanking the B2 gene. Panel a, the mutations introduced in the 5Ј and 3Ј flanks. Two constructs carrying either 5Ј or 3Ј mutations were established and transfected into wild-type cells. Panel b, total RNA from wild type (WT), the cell line carrying construct 3 as in A (lane 1), carrying the 5Ј mutation (lane 2), or the 3Ј mutation (lane 3) were subjected to primer extension analysis. The primer used in this experiment was as in A. C, RT-PCR analysis of tagged B2 transcript. Panel a, schematic representation of the construct with respect to the vector. The primers used in RT-PCR (panel b) are indicated, and the predicted length is given. Panel b, RT-PCR was performed as described under "Experimental Procedures" with oligonucleotides Neo and B2-tag-long. The PCR was analyzed on 1% agarose gel. Lanes 1-4, PCR with cDNA generated with anti-B2 tag oligonucleotide. Lanes 5-8, PCR with total RNA as a template, served as negative controls. Lanes 1 and 5, total RNA from the cell line carrying the 5Ј mutation. Lanes 2 and 6, total RNA from the cell line carrying the 3Ј mutation. Lanes 3 and 7, total RNA from cell line carrying construct 3 with wild-type flanking sequences (positive control). Lanes 4 and 8, total RNA from wild-type cells. (28,29). Recently, we compiled a large number of C/D snoR-NAs in both T. brucei and Trypanosoma cruzi genomes and found that with no exception these snoRNAs are organized in repeated clusters. All clusters that we identified carry both C/D and H/ACA RNAs. This genome organization resembles mostly the organization in plants, which also carry a cluster encoding for mixed C/D and H/ACA RNAs. In yeast, the clusters encode for only one type of snoRNA (6), and in metazoa such as mice, humans, and Drosophila, all snoRNAs are intronic, and each intron encodes for only a single snoRNA (9).
The discovery of the eight newly identified H/ACA RNAs in this study raises the possibility that, most, if not all, trypanosome H/ACA-like RNAs are composed of a single hairpin and carry the AGA box. Moreover, the 23 newly identified H/ACA RNAs from the T. brucei genome also carry a single hairpin and an AGA box at the 3Ј end, 2 suggesting that this is the prototype of the trypanosomatid H/ACA RNA. Another organism possessing single hairpin pseudouridine guides is archaea (36,37), whereas all eukaryotic H/ACA snoRNAs carry two hairpins connected by the hinge region. Interestingly, archaea were also shown to possess H/ACA-like RNA that carry multiple hairpins. We currently have no evidence of the existence of such RNAs in trypanosomatids. The structure of trypanosomes, archaea, and eukaryotic H/ACA RNA suggests that the single hairpin H/ACA RNA may have been the ancestor of multihairpin H/ACA RNAs that may have originated from the fusion of adjacent single hairpin RNAs. This hypothesis is supported by the fact that each of the two hairpins associates with one set of four core proteins (38). Based on the fact that in trypanosomatids almost all snoRNA genes are clustered and transcribed into polycistronic precursors, the maturation of individual snoRNAs should involve the endonucleolytic cleavage and exonucleolytic trimming, like the case of yeast polycistronic snoRNAs. In higher eukaryotes, most of the C/D box RNAs carry a short (4 -6 bp) stem structure formed between the 5Ј and 3Ј end coding sequences. This stem serves as a signal for pre-RNA processing (39,40). In trypanosomes, the flanking sequences of most snoRNAs have the potential to form a duplex (24,28), and it was shown that the flanking sequences are required for snoRNA accumulation (28). Indeed, mutational studies performed during this study indicate the importance of the first 10-bp flanking sequence for snoRNA processing, because in these mutants the polycistronic transcript was unable to produce a stable tagged B2 transcript. However, 15 bp of the 3Ј-flanking sequence was sufficient to obtain maximal expression of the tagged B2 gene, suggesting that the processing signals lie very close to the 3Ј end of the snoRNA. The duplex that is potentially formed between the 10-nt flanking sequence may serve as a signal for endonucleolytic cleavage, similar to pre-snoRNA processing by Rnt1 in yeast (6). Ongoing studies in our laboratory are attempting to identify the role of exosome components recently identified in T. brucei (41) in terms of their function in snoRNA precursor processing.
In eukaryotes, such as yeast and vertebrates, snoRNA genes are transcribed by distinct promoters. So far, there is no evidence of the presence of such elements in trypanosomes. Here we made use of the antisense silencing approach we discovered for snoRNA genes (31) to examine the ability of the 700-bp fragment present upstream from the snoRNA repeats to promote transcription of the snoRNA cluster.
As described previously and also illustrated in this study, the expression of antisense RNA to the tagged snoRNA gene eliminated the production of both the sense-tagged snoRNA transcript as well as the wild-type transcript. This silencing was FIG. 6. The effect of the upstream fragment cloned on the level of neo mRNA and the snoRNA-2 antisense transcript. A, 58-2 and 2-3 cell lines were selected with an elevated G418 level as indicated above the lanes (in g/ml). Total RNA was prepared from different cell lines and subjected to primer extension using primers 36815 and 43362, complementary to neo mRNA and h1 snoRNA, respectively. The extension products are marked and indicated. B, detection of the level of snoRNA-2 antisense transcript. The same RNA that was used in A was subjected to primer extension using a sense primer 22182 specific to snoRNA-2 that extends the antisense transcript. The product is marked and indicated. The numbers above the lanes indicate the concentration of G418 in which the cell lines were selected. WT, wild type.
FIG. 5. The ability of the 700-bp upstream sequence to enhance transcription. A, schematic representation of the constructs. The orientation of the tagged gene is marked with arrows, and the names of the constructs are given. The sites of restriction enzymes are indicated. pX-neo, the expression vector; h2, the h2 snoRNA; sno2, the snoRNA-2 RNA; up, the 700-bp sequence upstream from B2 repeats. B, primer extension analysis to determine the expression of the tagged snoRNA-2 gene. Primers used in the reaction are 16865 and 43362, complementary to the wild-type and tagged snoRNA-2, and h1 RNA, respectively. The extension products are marked with arrows and are indicated. Lane WT, total RNA from wild type; lanes 1-4, RNA from cell lines carrying the constructs 58-1, 58-2, 2-3, and 2-4, respectively. C, primer extension analysis to determine the level of h2 RNA using primers 22076 and 43388, specific to h2 and B5 snoRNA, respectively. The extension products are marked with arrows and are indicated. Lanes 1-4, RNA from 58-1, 58-2, 2-3, and 2-4 cell lines, respectively. M, marker was as described in Fig. 2. The sizes of the marker in nucleotides (nt) are indicated. accompanied by the production of siRNAs (31). However, we propose that the mechanism of silencing may differ from the conventional RNA i mechanism. The siRNAs we detected may have been generated from the cleavage of the double-stranded RNA produced from the sense and antisense transcript directly by RNase III (a Dicer-like enzyme) without the need for the RNA-induced silencing complex. Indeed, studies have recently provided evidence that the expression of a stem-loop structure in Leishmania species did not elicit degradation of the target mRNA (42), suggesting that Leishmania lacks the conventional RNA i machinery. Based on all the current data, we suggest that cleavage of the target snoRNA is mediated by RNase III through the formation of a double-stranded RNA duplex between the sense and antisense RNAs and that the siRNAs produced in this cleavage are not catalytic and cannot promote the cleavage of additional target RNAs. This is why very high levels of antisense RNA are needed to elicit silencing of snoRNA. The data presented here support our model of silencing via antisense RNA.
The transcription in the pX-neo vector is asymmetric and bi-directional, and the activity of transcription from the neo strand is much higher than from the other strand (31). However, the 700-bp element promotes the transcription from the otherwise poorly transcribed strand. The presence of the 700-bp element therefore enhanced promotion of this strand increasing the level of tagged snoRNA-2 transcript. In addition, because it is known that due to steric hindrance transcription cannot take place simultaneously on two strands, the tran-scription from the other stand encoding for the antisense snoRNA-2 transcript was most probably reduced (construct 2-3). As a result of increasing the snoRNA-2 transcript and decreasing the antisense to snoRNA-2 silencing of this RNA was dimmed. The 700-bp element acts only in an orientationdependent manner, because attenuation of silencing was detected only when this fragment was present in the transcription direction of the snoRNA but not in the opposite orientation (construct 2-4) (Fig. 5).
Identification of this 700-bp fragment as a potential promoter is not trivial. So far, all other trypanosome small RNA genes were shown to have their own promoters. The small nuclear RNAs and 7SL RNA genes are transcribed by polymerase III by an extragenic tRNA that serves as a promoter (43). The SL RNA gene is transcribed by polymerase II; its promoter as well as the transcription factors and their binding sites were characterized (21). However, proving the existence of RNA polymerase II promoters for protein-coding genes is very elusive. Nevertheless, there are reports to support the existence of such promoters (44,45). However, in Leishmania and Leptomonas it is possible to obtain expression of a marker from episomes containing only the plasmid backbone; the marker gene is preceded by a splice acceptor and a synthetic polypyrimidine tract but with no obvious promoter (46,47). However, research from the laboratory of P. Myler and K. Stuart (Seattle Biomedical Research Institute) supports the existence of promoters along the Leishmania chromosomes. Stably maintained episomes carrying domains from the inflection point that marks the bi-directional transcription chromosome I expressed up to 10-fold more protein from the reporter gene than analogous episomes lacking this region (23). In addition, nuclear run-on experiments on chromosome III also indicated that both promoters and terminators are present in regions where transcription changes direction and where the tRNA gene is found at the end of a transcription unit. Interestingly, as depicted in Fig. 1, we also identified a tRNA-like molecule at the 3Ј flank of the snoRNA cluster. Surprisingly, the different coding strand inflection points in Leishmania do not show any sequence similarity. All the data, however, support the existence of a few promoters per chromosome in Leishmania. However, these promoter regions do not share sequence similarity and may not be conventional TATA-containing promoters like promoters in other eukaryotes.
We propose that the 700-bp element that we identified here is one of the several "promoter-like" sequences present along the chromosome, because this sequence is able to enhance the expression of the snoRNA gene in an orientation-dependent manner. Interestingly, this fragment is rich in polypyrimidine tracts that are known to attract the splicing machinery (48,49). Because transcription and splicing are coupled, and the machinery of polymerase II is equipped with all the splicing and polyadenylation factors, this domain may recruit transcription factors via preferential binding of the splicing and polyadenylation factors. However, the presence of a transcript encoding for the 700-bp fragment does not allow us to define this region as a conventional promoter sequence, because normally promoter regions themselves are not transcribed. However, one can envision a mechanism for transcriptional regulation in these parasites that is assisted by potential elements along the long chromosome. These sequences may operate only in cases where polycistronic transcription originating from the upstream region ceased. These promoter-like sequences may therefore serve as reviving stations to rescue transcription along the chromosome, keeping the chromosome transcriptionally active. However, these elements may also have a special regulatory role when there is an immediate need to enhance transcription in order to support growth. For instance, snoR-NAs are in high demand when the rRNA synthesis level is elevated during accelerated growth. It is too simplistic to assume that transcription of half or more of an entire chromosome depends on initiation only from a single region present at the inflection point that marks the bi-directional transcription of the trypanosomatid chromosomes. It will therefore be of interest to examine other intergenic regions upstream from the first repeated genes for their ability to promote transcription.