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

Xu, Yuxin; Liu, Li; López-Estraño, Carlos; Michaeli, Shulamit

doi:10.1074/jbc.m007007200

Cited by 37 publications

(73 citation statements)

References 50 publications

(74 reference statements)

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“…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.…”

Section: Identification Of Two Clusters Encoding For Both C/d and H/amentioning

confidence: 99%

“…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.…”

Section: Oligonucleotidesmentioning

confidence: 99%

“…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 snoRNAs in eukaryotes and archaea, the trypanosome H/ACA RNAs possess unique features.…”

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confidence: 99%

“…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).…”

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Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

Liang

Ochaion

et al. 2004

Journal of Biological Chemistry

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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...

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Section: Identification Of Two Clusters Encoding For Both C/d and H/amentioning

confidence: 99%

Section: Oligonucleotidesmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

Liang

Ochaion

et al. 2004

Journal of Biological Chemistry

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“…Primer Extension Analysis-Total RNA (10 g) from induced and uninduced cells was subjected to primer extension analysis as described (36). 5Ј end-labeled oligonucleotides were used in the extensions to detect the level of U1, U2, U4, U5, U6, SLA1, and SL RNA.…”

Section: Constructs and Establishment Of Stable Cellmentioning

confidence: 99%

Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei

Liu¹,

Liang²,

Uliel

et al. 2004

Journal of Biological Chemistry

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RNA interference of Sm proteins inTrypanosoma brucei demonstrated that the stability of the small nuclear RNAs (U1, U2, U4, U5) and the spliced leader RNA, but not U6 RNA, were affected upon Sm depletion (Mandelboim, M., Barth, S., Biton, M., Liang, X. H., and Michaeli, S. (2003) J. Biol. Chem. 278, 51469-51478), suggesting that Lsm proteins that bind and stabilize U6 RNA in other eukaryotes should exist in trypanosomes. In this study, we identified seven Lsm proteins (Lsm2p to Lsm8p) and examined the function of Lsm3p and Lsm8p by RNA interference silencing. Both Lsm proteins were found to be essential for U6 stability and mRNA decay. Silencing was lethal, and cisand trans-splicing were inhibited. Importantly, silencing also affected the level of U4.U6 and the U4.U6/U5 tri-small nuclear ribonucleoprotein complexes. The presence of Lsm proteins in trypanosomes that diverged early in the eukaryotic lineage suggests that these proteins are highly conserved in both structure and function among eukaryotes. Interestingly, however, Lsm1p that is specific to the mRNA decay complex was not identified in the genome data base of any kinetoplastidae, and the Lsm8p that in other eukaryotes exclusively functions in U6 stability was found to function in trypanosomes also in mRNA decay. These data therefore suggest that in trypanosomes only a single Lsm complex may exist.

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Small Nucleolar RNAs: Identification, Structure, and Function

Söderbom

2006

Small RNAs

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

Cited by 37 publications

References 50 publications

Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

Small Nucleolar RNA Clusters in Trypanosomatid Leptomonas collosoma

Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei

Small Nucleolar RNAs: Identification, Structure, and Function

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