Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Mottram, Jeremy C.; Perry, Karen L; Lizardi, Paul M.; Lührmann, Reinhard; Agabian, Nina; Nelson, Richard G.

doi:10.1128/mcb.9.3.1212

Cited by 117 publications

(133 citation statements)

References 60 publications

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“…'C for 1 h and autorachographed [6] fig.2A and B, lanes 1 and 4), a >23 kb doublet for the T. brucei &rnHl digest (lane 6) and a single >23 kb band for T. cruzi (lane 3). A T. brucei cDNA library in Xgt 11 was also screened and several clones were isolated.…”

Section: Methodsmentioning

confidence: 99%

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C‐terminal extension

et al. 1989

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of molecular and glo~og~~al Sctences, ~~lverslty of Strrtmg, Stn-lmg FKP 4LA and ~De~art~nt of Zoology, ~~~vers~ty of Gl~gow, Glasgow GIL? 8QQ, Scotland Received 7 September 1989, revtsed version recetved 26 September 1989 A cDNA for a Trypanosoma bnrcer cysteme protemase has been cloned and sequenced The deduced protem can be chuded mto four domains, based on homologies with other cysteme protemases the pre-, pro-and central relpons show considerable homology to the cathepsrn L class of mammahan enzymes, whilst the long C-terminal extension chstmgmshes the trypanosome enzyme from all mammalian cysteme protanases reported This 108 ammo acid extension, wluch includes 9 contiguous prohnes near the Junctron with the central domain, appears likely to be processed m part to produce the mature enzyme, and may be mvolved m targettmg the protein wrthm the cell The trypanosome genome contams more than 20 copies of the cysteme protemase gene arranged m a long tandem array Trypanosome, Cysteme protemase 1, INTRODUCTIONThe cysteine proteinases (EC 3.4.22) are proving a valuable group for investigating the relationship between enzyme structure and function. Mammalian lysosomal cathepsins (B, H, L) and plant proteinases such as papain have been the most extensively studied, but similar enzymes occur widely in invertebrates and protozoa [l]. Notably, they occur at high activity in a number of parasitic protozoa, many of which contain multiple enzymes that in some species are developmentally regulated [2,3]. The abundance of cysteine proteinases in a variety of parasites, their potential role in the host-parasite interaction and virulence, and the advent of families of specific irreversible inhibitors [4], have made these enzymes attractive targets for chemotherapeutic attack. fig.2A and B, lanes 1 and 4), a >23 kb doublet for the T. brucei &rnHl digest (lane 6) and a single >23 kb band for T. cruzi (lane 3). A T. brucei cDNA library in Xgt 11 was also screened and several clones were isolated. The largest insert was subcloned into Bluescript plasmid (pTCP-Fl).On genomic Southerns, the cDNA hybridised to the same fragments as the oligonucleotides for the Hind1 and BamHl digests, with an extra band present in the Pstl digest ( fig.2C). Although the oligonucleotides hybridised to DNA of both trypanosomes with equal intensity, the T. bmcei cDNA hybridised only weakly to the T.

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Section: Methodsmentioning

confidence: 99%

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C‐terminal extension

et al. 1989

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“…Like in cis-splicing, U2, U4, and U6 snRNAs have been characterized in trypanosomes and were shown to be essential for trans-splicing (4). However, despite their essential role in cis-splicing, U1 or U5 have not been identified (5). Without exception, all trypanosome small nuclear RNAs (snRNAs) have been shown to be shorter than their cis-splicing counterparts.…”

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The U5 RNA of trypanosomes deviates from the canonical U5 RNA: The Leptomonas collosoma U5 RNA and its coding gene

Ben-Shlomo²,

Michaeli³

1997

Proc. Natl. Acad. Sci. U.S.A.

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Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5 phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016-4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4͞U6⅐U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem-loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNA Cys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5 splice site region (positions ؊1 and ؉1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis-and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.Trans-splicing was first discovered in trypanosomes as a novel RNA processing pathway linking a common 39-nt spliced leader (SL) to all mRNAs (1). Trans-splicing is a two-step transesterification reaction that involves two substrates, the SL RNA and a 3Ј acceptor pre-mRNA. In the reaction, the two introns derived from the SL RNA and pre-mRNA form the Y-branched intermediate, analogous to the intron lariat intermediate of cis-splicing. The final product is an mRNA carrying the spliced leader at the 5Ј end (1). This process has been shown to occur in a number of other organisms, such as nematodes, trematodes and Euglena, in conjunction with cissplicing, whereas in kinetoplastidae it is the only RNA processing pathway (2).In cis-splicing the small ribonucleoproteins (RNPs) play a dual role in juxtaposing the splice sites and in contributing part of the catalytic center (3). Like in cis-splicing, U2, U4, and U6 snRNAs have been characterized in trypanosomes and were shown to be essential for trans-splicing (4). However, despite their essential role in cis-splicing, U1 or U5 have not been identified (5). Without exception, all trypanosome small nuclear RNAs (snRNAs) have been shown to be shorter than their cis-splicing counterparts. The presence of a uniform 5Ј splice site carried on the SL RNA may have changed the requirement for U1 RNA in trans-splicing. The ability of the SL RNA to substitute for the need of U1 RNA in cis-splicing supports the idea that SL RNA may bear some of the critical functions of U1 RNP (6)....

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“…Trypanosome SL RNA, U1, U2, U4, and U5 RNAs associate with Smcore proteins (28 -31). The Sm binding sites on the snRNAs were found to be divergent compared with the canonical site (32). The first Sm protein was identified as an SL RNP core protein in Leptomonas collosoma (29).…”

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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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Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Cited by 117 publications

References 60 publications

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C‐terminal extension

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C‐terminal extension

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: The Leptomonas collosoma U5 RNA and its coding gene

Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei

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