Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes.

Nakaar, Valerian; Daré, Amos O.; Hong, David S.; Ullu, Elisabetta; Tschudi, Christian

doi:10.1128/mcb.14.10.6736

Cited by 79 publications

(68 citation statements)

References 31 publications

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“…tRNA genes that are divergently transcribed relative to the small RNA genes, were found upstream to the U3, U6, and 7SL RNAs (14,30) and to U5 RNA (this study), suggesting that this transcriptional regulation is related neither to the cap structure of these RNAs nor to the identity of their binding proteins or even to their cellular compartmentalization. This genomic organization may therefore reflect a very archaic mode of transcriptional regulation with a potential to coordinate the synthesis of small RNAs that are essential for growth.…”

Section: Discussionmentioning

confidence: 88%

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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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Section: Discussionmentioning

confidence: 88%

“…The existence of a tRNA gene 98 nt upstream to the U5 that is encoded by the opposite strand may suggest that the U5 gene belongs to the same transcriptional regulation unit as the U3, U6, and 7SL RNA genes that have been shown to be transcribed by RNA polymerase III (14).…”

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confidence: 99%

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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“…The U6 small nuclear (sn) RNA gene promoter contains three elements: one located within the 5Ј-portion of the intragenic region and two located within an upstream, but inversely oriented, tRNA gene. The two intragenic tRNA promoter elements, called A and B boxes, cofunction in both U6 and tRNA expression (9). Two abundant cell surface proteins in the African trypanosome Trypanosoma brucei are encoded by genes with promoter elements that resemble RNA pol I promoters in both structure and ␣-amanitin resistance.…”

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confidence: 99%

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

Luo

Gilinger

Mukherjee

et al. 1999

Journal of Biological Chemistry

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Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m 7 G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical doublestranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (؉1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.Molecular studies of trypanosomatids, a ubiquitous and diverse family of protozoan pathogens, have revealed strikingly unusual mechanisms of mRNA synthesis. One central device is that two independent transcription events direct each mRNA produced in the trypanosome nucleus (for review, see Ref. 1). The protein-coding portion is transcribed as a single primary mRNA, often containing several open reading frames flanked by 5Ј-and 3Ј-untranslated regions. The capped 5Ј-end portion is transcribed as a short spliced leader (SL) 1 RNA. The two parts are fused in a trans-splicing reaction that yields a functional mRNA. During fusion, the 39 nt present on the 5Ј-end of the SL RNA (and referred to as the SL) are transferred to a region upstream from the coding region on the primary mRNA (2). Addition of the SL provides each mRNA with an m 7 G cap as well as four extensively methylated nucleotides, at positions 1-4 within the 39-nt SL RNA (3).The SL RNA is transcribed from a highly reiterated set of genes. In contrast to the long primary transcripts that form the bulk of the mature mRNA, each SL RNA has a discrete transcriptional start site. ␣-Amanitin studies show that it is very probable, though not proven, that the SL RNA gene is transcribed by RNA polymerase (pol) II. The primary SL RNA transcript and the transcript present in the trans-splicing spliceosome possess identical 5Ј-and 3Ј-ends, indicating that both transcription initiation and termination regulate the accumulation of SL RNA. SL RNA expression has been monitored using independe...

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“…The U1, U2, U4, and U5 that are transcribed by RNA polymerase II bind Sm proteins, whereas the U6 that binds to Lsm proteins is transcribed by RNA polymerase III (1). However, in trypanosomes all the U snRNAs are transcribed by polymerase III (27). The SL RNA, however, is transcribed by RNA polymerase II and also binds to Sm proteins (40).…”

Section: Discussionmentioning

confidence: 99%

“…All spliceosomal U snRNAs were identified in this organism (24). As opposed to their eukaryotic counterparts, the trypanosome U snRNAs are transcribed by RNA polymerase III (27). Trypanosome SL RNA, U1, U2, U4, and U5 RNAs associate with Smcore proteins (28 -31).…”

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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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Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes.

Cited by 79 publications

References 31 publications

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

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

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei

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