Affinity purification of Trypanosoma brucei small nuclear ribonucleoproteins reveals common and specific protein components.

Pálfi, Zsófia; Günzl, Arthur; Cross, Mike; Bindereif, Albrecht

doi:10.1073/pnas.88.20.9097

Cited by 61 publications

(76 citation statements)

References 27 publications

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“…In other eukaryotes, a strong correlation exists between the TMG cap, binding of Sm proteins, and the polymerase that transcribes the RNA. In trypanosomes, such stringent rules do not exist as the core proteins are apparently common to all spliceosomal RNAs (28), even though these RNAs are transcribed by different polymerases-i.e., polymerase II for the SL RNA (29), and polymerase III for U2 and U6 (30). In addition, core proteins bind RNAs that harbor different cap structures: the U2 and U4 are TMG capped (5), the SL RNA possesses a 7 metG hypermodified ''cap 4'' (31), and the SLA (8) and the U5 RNA (ref.…”

Section: Discussionmentioning

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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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: 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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“…Capping of mRNAs by SL sequence through trans-splicing is a unique RNA maturation process characteristic of Trypanosoma subspecies. Although different than cis-splicing, trans-splicing in T. brucei still requires canonical spliceosomal U RNA machinery [57] and Sm component proteins [58,59]. Telomerase RNA maturation in fission yeast involves mechanism akin to cis-splicing [60], which requires sequential binding of Sm proteins to telomerase RNA [61].…”

Section: Discussionmentioning

confidence: 99%

A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

et al. 2013

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Telomerase is a ribonucleoprotein enzyme typically required for sustained cell proliferation. Although both telomerase activity and the telomerase catalytic protein component, TbTERT, have been identified in the eukaryotic pathogen Trypanosoma brucei, the RNA molecule that dictates telomere synthesis remains unknown. Here, we identify the RNA component of Trypanosoma brucei telomerase, TbTR, and provide phylogenetic and in vivo evidence for TbTR's native folding and activity. We show that TbTR is processed through trans-splicing, and is a capped transcript that interacts and copurifies with TbTERT in vivo. Deletion of TbTR caused progressive shortening of telomeres at a rate of 3-5 bp/population doubling (PD), which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner. Remarkably, introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences, demonstrating that telomere synthesis in T. brucei is dependent on TbTR. We also propose a secondary structure model for TbTR based on phylogenetic analysis and chemical probing experiments, thus defining TbTR domains that may have important functional implications in telomere synthesis. Identification and characterization of TbTR not only provide important insights into T. brucei telomere functions, which have been shown to play important roles in T. brucei pathogenesis, but also offer T. brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes.

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“…Antisense affinity selection can purify snRNPs (or other RNPs) sufficiently for the associated proteins to be analysed directly [3&32]. This has been particularly useful for studies of snRNP proteins in trypanosome extracts, where no antibodies were available that would recognise the trypanosome snRNPs [31].…”

Section: Febs Letters June 1993mentioning

confidence: 99%

Antisense oligonucleotides made of 2'‐O‐alkylRNA: their properties and applications in RNA biochemistry

1993

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Oligo(2'-0-alkylribonucleotides) have been developed recently as novel oligonucleotide analogues with properties that enhance their use as antisense probes. They possess high chemical stability and are resistant to hydrolysis by DNA-or RNA-specific nucleases. Many forms of oligo(2'-Oalkyhibonucleotides) hybrid& specifically and efficiently to complementary RNA sequences, forming stable duplexes that are not substrates for cleavage by RNase H. In combination with prosthetic reporter groups, such as biotin, DNP or fluorophores, oligo(Y-0-alkylribonucleotides) have important applications in a wide range of biochemical studies on RNA function and structure.

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Affinity purification of Trypanosoma brucei small nuclear ribonucleoproteins reveals common and specific protein components.

Cited by 61 publications

References 27 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

A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

Antisense oligonucleotides made of 2'‐O‐alkylRNA: their properties and applications in RNA biochemistry

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