Artigo Acesso aberto Revisado por pares

Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês

10.1074/jbc.m308264200

ISSN

1083-351X

Autores

Xue‐hai Liang, Avivit Ochaion, Yuxin Xu, Qing Liu, Shulamit Michaeli,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

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. 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. In eukaryotes, pre-rRNA transcript undergoes numerous site-specific modifications before cleavage. Two prevalent modifications, 2′-O-methylation and conversion of uridine to pseudouridine, are guided by C/D box and H/ACA box small nucleolar RNAs (snoRNAs), 1The abbreviations used are: snoRNAsmall nucleolar RNASL RNAspliced leader RNASLAspliced leader associated RNAsiRNAsmall interfering RNAORFopen reading framentnucleotideRTreverse transcriptase.1The abbreviations used are: snoRNAsmall nucleolar RNASL RNAspliced leader RNASLAspliced leader associated RNAsiRNAsmall interfering RNAORFopen reading framentnucleotideRTreverse transcriptase. respectively (1Kiss-László Z. Henry Y. Bachellerie J.P. Caizergues-Ferrer M. Kiss T. Cell. 1996; 85: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 2Ni J. Tien A.L. Fournier M.J. Cell. 1997; 89: 565-573Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 3Ganot P. Bortolin M.L. Kiss T. Cell. 1997; 89: 799-809Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). The C/D snoRNAs carry short conserved motives, as do the C box (RUG-AUGA) and D box (CUGA), near the 5′ and 3′ ends, respectively, as well as the internal D′ and C′ boxes. Sequences (>10 nt) upstream from the D and/or D′ box can interact with the target RNA by perfect base pairing. The 5th nt on the target RNA upstream from the D or D′ box in the duplex is methylated, which is known as the “+5 rule” (1Kiss-László Z. Henry Y. Bachellerie J.P. Caizergues-Ferrer M. Kiss T. Cell. 1996; 85: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). 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 3′ 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 (2Ni J. Tien A.L. Fournier M.J. Cell. 1997; 89: 565-573Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 3Ganot P. Bortolin M.L. Kiss T. Cell. 1997; 89: 799-809Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). small nucleolar RNA spliced leader RNA spliced leader associated RNA small interfering RNA open reading frame nucleotide reverse transcriptase. small nucleolar RNA spliced leader RNA spliced leader associated RNA small interfering RNA open reading frame nucleotide reverse transcriptase. In vertebrates, all guide snoRNAs are intronic (4Terns M. Terns R. Gene Expr. 2002; 10: 17-39PubMed Google Scholar, 5Weinstein L.B. Steitz J.A. Curr. Opin. Cell Biol. 1999; 11: 378-384Crossref PubMed Scopus (248) Google Scholar). Most yeast snoRNAs are encoded by independent genes, some of which are found in clusters. However, seven intronic genes were also described (6Qu L.H. Henras A. Lu Y.J. Zhou H. Zhou W.X. Zhu Y.Q. Zhao J. Henry Y. Caizergues-Ferrer M. Bachellerie J.P. Mol. Cell. Biol. 1999; 19: 1144-1158Crossref PubMed Scopus (136) Google Scholar). In plants, most snoRNAs are clustered and are independently transcribed, but intronic snoRNA gene clusters also exist (7Brown J. Clark G. Leader D. Simpson C. Lowe T. RNA (New York). 2001; 7: 1817-1832PubMed Google Scholar, 8Liang D. Zhou H. Zhang P. Chen Y.Q. Chen X. Chen C.L. Qu L.H. Nucleic Acids Res. 2002; 30: 3262-3272Crossref PubMed Scopus (35) Google Scholar, 9Brown J.W. Echeverria M. Qu L.H. Trends Plant Sci. 2003; 8: 42-49Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The intronic snoRNA genes are transcribed from the promoter of host genes by RNA polymerase II (10Kiss T. EMBO J. 2001; 20: 3617-3622Crossref PubMed Scopus (341) Google Scholar, 11Kiss T. Cell. 2002; 109: 145-148Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). 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 (6Qu L.H. Henras A. Lu Y.J. Zhou H. Zhou W.X. Zhu Y.Q. Zhao J. Henry Y. Caizergues-Ferrer M. Bachellerie J.P. Mol. Cell. Biol. 1999; 19: 1144-1158Crossref PubMed Scopus (136) Google Scholar). In plants, promoters expressing polycistronic snoRNA clusters have not yet been identified. In vertebrates and yeast, only a single snoRNA exists per intron. The maturation of intron-encoded snoRNAs involves largely splicing-dependent processing by exonucleases from linearized and debranched lariats (12Filipowicz W. Pogacic V. Curr. Opin. Cell Biol. 2002; 14: 319-327Crossref PubMed Scopus (319) Google Scholar). In plants, the processing of intronic snoRNA clusters requires endonucleolytic activity (9Brown J.W. Echeverria M. Qu L.H. Trends Plant Sci. 2003; 8: 42-49Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The yeast polycistronic snoRNA precursors are processed by Rnt1p, a homologue of bacterial RNase III (13Chanfreau G. Legrain P. Jacquier A. J. Mol. Biol. 1998; 284: 975-988Crossref PubMed Scopus (138) Google Scholar). The liberated 5′ and 3′ ends are further processed by 5′ → 3′-exonucleases Rat1p and Xrn1p, and the 3′ → 5′-exosome (including Rrp6p) (14Allmang C. Kufel J. Chanfreau G. Mitchell P. Petfalski E. Tollervey D. EMBO J. 1999; 18: 5399-5410Crossref PubMed Scopus (486) Google Scholar, 15van Hoof A. Lennertz P. Parker R. Mol. Cell. Biol. 2000; 20: 441-452Crossref PubMed Scopus (296) Google Scholar). 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 (16Agabian N. Cell. 1990; 61: 1157-1160Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 17Simpson L. Sbicego S. Aphasizhev R. RNA (New York). 2003; 9: 265-276Crossref PubMed Scopus (137) Google Scholar). 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 trans-splicing and polyadenylation (18Huang J. van der Ploeg L.H. Mol. Cell. Biol. 1991; 11: 3180-3190Crossref PubMed Scopus (53) Google Scholar, 19Ullu E. Matthews K.R. Tschudi C. Mol. Cell. Biol. 1993; 13: 720-725Crossref PubMed Scopus (152) Google Scholar). 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 (20Myler P.J. Sisk E. McDonagh P.D. Martinez-Calvillo S. Schnaufer A. Sunkin S.M. Yan S. Madhubala R. Ivens A. Stuart K. Biochem. Soc. Trans. 2000; 28: 527-531Crossref PubMed Scopus (44) Google Scholar). So far, there is no evidence of the existence of a polymerase II promoter that transcribes protein-coding 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 (21Campbell D.A. Sturm N.R. Yu M.C. Parasitol. Today. 2000; 16: 78-82Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Two protein-coding genes, the Trypanosoma brucei variant surface glycoprotein and the EP (procyclin), are transcribed by RNA polymerase I (22Lee M.G. Van der Ploeg L.H. Annu. Rev. Microbiol. 1997; 51: 463-489Crossref PubMed Scopus (91) Google Scholar). 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 (23Clayton C.E. EMBO J. 2002; 21: 1881-1888Crossref PubMed Scopus (444) Google Scholar). The probable presence of gene clusters carrying both C/D and H/ACA-like RNAs has been reported in studying snoRNA genes in trypanosomatids (24Liang X. Liu L. Michaeli S. J. Biol. Chem. 2001; 276: 40313-40318Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The first C/D snoRNA identified in Leptomonas collosoma was termed snoRNA-2 and was shown to obey the +5 methylation guiding rule (25Levitan A. Xu Y. Ben-Dov C. Ben-Shlomo H. Zhang Y. Michaeli S. Nucleic Acids Res. 1998; 26: 1775-1783Crossref PubMed Scopus (21) Google Scholar). Additional C/D snoRNAs were identified in T. brucei by immunoprecipitation with anti-fibrillarin antibody (26Dunbar D.A. Wormsley S. Lowe T.M. Baserga S.J. J. Biol. Chem. 2000; 275: 14767-14776Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). C/D snoRNAs were also found in a locus encoding for the spliced leader-associated RNA (SLA1 RNA) gene (26Dunbar D.A. Wormsley S. Lowe T.M. Baserga S.J. J. Biol. Chem. 2000; 275: 14767-14776Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 27Roberts T. Dungan J. Watkins K. Agabian N. Mol. Biochem. Parasitol. 1996; 83: 163-174Crossref PubMed Scopus (21) Google Scholar). Transcription analysis in L. collosoma and T. brucei suggests that snoRNAs are transcribed as polycistronic RNAs by polymerase II (28Xu Y. Liu L. Lopez-Estraño C. Michaeli S. J. Biol. Chem. 2001; 276: 14289-14298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 29Dunbar D.A. Chen A.A. Wormsley S. Baserga S.J. Nucleic Acids Res. 2000; 28: 2855-2861Crossref PubMed Google Scholar). The snoRNA genes are organized as reiterated gene clusters that are repeated more than five times in the genome (25Levitan A. Xu Y. Ben-Dov C. Ben-Shlomo H. Zhang Y. Michaeli S. Nucleic Acids Res. 1998; 26: 1775-1783Crossref PubMed Scopus (21) Google Scholar). 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 (28Xu Y. Liu L. Lopez-Estraño C. Michaeli S. J. Biol. Chem. 2001; 276: 14289-14298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Although the trypanosome C/D snoRNAs exactly fit the prototype C/D snoRNAs 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 (24Liang X. Liu L. Michaeli S. J. Biol. Chem. 2001; 276: 40313-40318Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). h1 has an AGA box but not an ACA box at the 3′ end. Recently, SLA1 was identified as a guide RNA that directs pseudouridylation at position –12 (relative to the 5′ splice site) on the SL RNA. Like h1, SLA1 also carries a single hairpin and possesses an AGA box at the 3′ end (30Liang X.H. Xu Y.X. Michaeli S. RNA (New York). 2002; 8: 237-246Crossref PubMed Scopus (55) Google Scholar). 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 RNAi 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 RNA-induced 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 (31Liang X.H. Liu Q. Michaeli S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7521-7526Crossref PubMed Scopus (37) Google Scholar). 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 3′ 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 orientation-dependent 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. Oligonucleotides—The oligonucleotides used are as follows. B2:26553, 5′-CGGGATCCCCGTTGCGCTATTCGGTCTT-3′, sense, carrying a BamHI site, is specific for the B2 upstream flanking sequence, from position –63 to –82. 35178, 5′-CGGGATCCCAGCCGCACGTCGCG-3′, antisense, carrying a BamHI site, is complementary to the downstream flanking sequence of B2, from position 93 to 108. 35758, 5′-CGGGATCCAACGCGCACAGGCC-3′, antisense, carrying a BamHI site, is complementary to the downstream flanking sequence of B2, from position 114 to 127. 35759, 5′-CGGGATCCCGCAGGTACGCAG-3′, antisense, carrying a BamHI site, is complementary to the downstream flanking sequence of B2, from position 155 to 167. 35179, 5′-CGGGATCCGCAGGTACGCGCATC-3′, antisense, carrying a BamHI site, is complementary to the downstream flanking sequence of B2, from position 128 to 142. 35176, 5′-CGGGATCCGTGCATGATGAGATA-3′, sense, carrying a BamHI site, is specific to the B2 coding region, from position 1 to 13. 26554, 5′-CGGGATCCCAGGTACGCAGGTACGCAGG-3′, antisense, carrying a BamHI site, is complementary to the downstream flanking sequence of B2, from position 184 to 203. 3′15, 5′-GCTCTAGACAGCCGCACGTCGCG-3′, antisense, carrying an XbaI site, is complementary to the downstream flanking sequence of B2, from position 93 to 108. 3′35, 5′-GCTCTAGAAACGCGCACAGGCC-3′, antisense, carrying an XbaI site, is complementary to the downstream flanking sequence of B2, from position 114 to 127. 41482, 5′-TATCTCATCATGCACTTTATATATTCACCGCAACACCGCA-3′, antisense, carrying 10-nt insertion, is complementary to the 5′ junction of B2, from position –25 to 15. 41485, 5′-CAGGCCACAGCACAGTTTATTTATTATGTCGTCAGAGTACGAAT-3′, antisense, carrying 10-nt insertion, is complementary to the 3′ junction of B2, from position 78 to 121. 20406, 5′-TTTCACATGCACGAGCATCC-3′, antisense, is complementary to B2 snoRNA, from position 35 to 54. Long B2 antitag, 5′-GCATCCGAATTCAAAGGT-3′, antisense, carrying the tagged sequence, is complementary to B2 snoRNA, from position 30 to 41. Neo, 5′-GTGCCTGCGTGACGG-3′, sense, is specific to the sequence in the vector, from position 1100 to 1115. Sno-2: Up5′ X, 5′-GCTCTAGATCATATTCCCCATTGCCGC-3′, sense, containing a XbaI site, is specific to the upstream sequence of B2 repeats, from position 450 to 469. UpA+X, 5′-GCTCTAGACACTAGCGGTGAGCAGTGGA-3′, antisense, containing an XbaI site, is complementary to the upstream sequence of B2 repeats, from position 1101 to 1120. 16865, 5′-CATCAGATGCCGGTAGTC-3′, antisense, is complementary to the snoRNA-2 gene, from position 67 to 83. 43362, 5′-CAATCTTGCACAGTGTCG-3′, antisense, is complementary to h1 snoRNA, from position 52 to 69. 22182, 5′-ACGTTCTGCAATCTGACCGCG-3′, sense, is specific to snoRA-2 snoRNA, from position 18 to 39, containing a T deletion in the middle. 36815, 5′-GAGGGAGGAATGAGGTGAGC-3′, antisense, is complementary to neo/hygro mRNA, from position 4996 to 5016 on the pX vector. 19208, 5′-CCGAGTATCGCCAAGG-3′, antisense, is complementary to h5 snoRNA, from position 21 to 37. h3-3A, 5′-CACTCTTTGGGGCGTTTC-3′, antisense, is complementary to h3 snoRNA, from position 47 to 64. 22076, 5′-CGGGATCCTGCCAGAATTGTCCCGTGC-3′, antisense, containing a BamHI site, is complementary to h2 snoRNA, from position 43 to 62. 43388, 5′-ACAGCTACCGCGAGTTGC-3′, antisense, is complementary to B5 snoRNA, from position 59 to 76. upss-1s, 5′-TGCGGACGGCTACGACGA-3′, sense, is specific to the upstream ORF, from 361 to 378. upss-1A, 5′-GGAACGGAGGTGGGGAAG-3′, antisense, is complementary to the intergenic region, from position 653 to 660. upss-2s, 5′-CCCCTTCGCTGCCACCAA-3′, sense, is specific to the intergenic region, from position 1050 to 1067. Plasmid Construction—B2 constructs were generated as described previously (28Xu Y. Liu L. Lopez-Estraño C. Michaeli S. J. Biol. Chem. 2001; 276: 14289-14298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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 3′-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 (28Xu Y. Liu L. Lopez-Estraño C. Michaeli S. J. Biol. Chem. 2001; 276: 14289-14298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) 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 5′ and 3′ mutations, respectively. The first-step PCR products were used as a megaprimer to amplify the full-length gene by second-step 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 5′ and 3′ 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 (25Levitan A. Xu Y. Ben-Dov C. Ben-Shlomo H. Zhang Y. Michaeli S. Nucleic Acids Res. 1998; 26: 1775-1783Crossref PubMed Scopus (21) Google Scholar). 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 5′ end with [γ-32P]ATP, as described previously (32Xu Y. Ben-Shlomo H. Michaeli S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8473-8478Crossref PubMed Scopus (45) Google Scholar). Subsequently, the RNA probe was used to screen the λ-phage genomic library (33Goldring A. Michaeli S. Gene (Amst.). 1995; 156: 139-144Crossref PubMed Scopus (16) Google Scholar). Clean λ DNA was digested with Sau3A and subcloned into the BamHI site of pBluescript KS+. Two clones obtained from two different λ 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 (24Liang X. Liu L. Michaeli S. J. Biol. Chem. 2001; 276: 40313-40318Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), which contains the entire repeat of the B2 snoRNA cluster. λ 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 KS+ 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 (34Goldring A. Zimmer Y. Ben-Yehuda E. Goncharov I. Michaeli S. Exp. Parasitol. 1996; 84: 28-41Crossref PubMed Scopus (17) Google Scholar). The pX-neo derived constructs were transfected into L. collosoma wild-type cells, as described previously (34Goldring A. Zimmer Y. Ben-Yehuda E. Goncharov I. Michaeli S. Exp. Parasitol. 1996; 84: 28-41Crossref PubMed Scopus (17) Google Scholar). 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 μg of total RNA was annealed with 50,000 cpm end-labeled primer(s) at 55–60 °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 μg 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 2× 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 snoRNAs, 70–90-nt RNAs obtained from the flow-through fraction of a DEAE-Sephacel column (32Xu Y. Ben-Shlomo H. Michaeli S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8473-8478Crossref PubMed Scopus (45) Google Scholar) were separated from the total RNA and recovered. Next, the eluted RNA was dephosphorylated and labeled with [γ-32P]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 λ 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 (24Liang X. Liu L. Michaeli S. J. Biol. Chem. 2001; 276: 40313-40318Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 25Levitan A. Xu Y. Ben-Dov C. Ben-Shlomo H. Zhang Y. Michaeli S. Nucleic Acids Res. 1998; 26: 1775-1783Crossref PubMed Scopus (21) Google Scholar, 28Xu Y. Liu L. Lopez-Estraño C. Michaeli S. J. Biol. Chem. 2001; 276: 14289-14298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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 3′ 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 (2Ni J. Tien A.L. Fournier M.J. Cell. 1997; 89: 565-573Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 3Ganot P. Bortolin M.L. Kiss T. Cell. 1997; 89: 799-809Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). 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 3′ 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 5′ and 3′ sequences are essential for proper expression. Deletion of the entire 5′ 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 3′ 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 (28Xu Y. Liu L. Lopez-Estraño C. Michaeli S. J. Biol. Chem. 2001; 276: 14289-14298Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In this study, we examined the expression of a snoRNA gene embedded within a cluster

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