Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

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

doi:10.1128/mcb.11.11.5516

Cited by 53 publications

(65 citation statements)

References 34 publications

(52 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The only two spliceosomal RNAs that possess identical motifs are SL and U5 RNAs. This motif is always present in a single-stranded region that was previously implicated in binding the T. brucei snRNP core proteins (24).…”

Section: Resultsmentioning

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.

View full text Add to dashboard Cite

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)....

show abstract

Section: Resultsmentioning

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.

View full text Add to dashboard Cite

show abstract

“…The hybridization analysis with U4-and U6-specific probes confirmed the presence of a U6 and a U4/U6 snRNP in extract (11). In total RNA, U4 was predominantly in the form of a U4/U6 RNA hybrid; only a very small fraction of U4 ran at the position of free U4 RNA; about one-third of U6, however, behaved as free U6 RNA; the rest of U4 and U6 RNAs comigrated and most likely represent the U4/U6 RNA hybrid (compare Fig.…”

Section: Methodsmentioning

confidence: 94%

“…In addition, the loop position G119 strongly interfered when modified and is conserved only in trypanosomatid U2 RNAs but not in U2 RNAs from cis-splicing systems (Fig. 8) (11). Since the SL RNP and the U2 and U4/U6 snRNPs contain, in addition to several snRNPspecific proteins, a common set of polypeptides (38) (33,38).…”

Section: Methodsmentioning

confidence: 99%

“…RESULTS trans-spliceosomal snRNPs contain stable core structures. snRNPs present in extracts of T. brucei have previously been identified by glycerol gradient sedimentation and by isopycnic centrifugation (11,33). To analyze trypanosomal snRNPs under native conditions in a manner that allows the direct comparison of multiple samples, we separated RNP complexes from T. brucei extracts by native gel electrophoresis; snRNP complexes were detected after electrophoretic transfer by probing with 32P-labeled antisense-snRNA oligonucleotides.…”

Section: Methodsmentioning

confidence: 99%

“…Cultures and extracts of the procyclic form of T. brucei brucei strain 427 were prepared as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.

Günzl¹,

Cross²,

Bindereif³

1992

Mol. Cell. Biol.

Self Cite

View full text Add to dashboard Cite

Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis-and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.All protein-coding mRNAs analyzed in trypanosomes to date arise by trans splicing of the 140-nucleotide spliced leader RNA (SL RNA), which contributes the common, 40-nucleotide spliced leader, and a precursor RNA with the protein-coding exon. The chemistry of cleavage-ligation reactions follows the same principles in both cis and trans splicing, with small nuclear ribonucleoproteins (snRNPs) functioning as essential cofactors (51). Trypanosomatids appear to process mRNAs exclusively through trans splicing, whereas in nematodes both cis and trans splicing can occur within the same precursor RNA (reviewed in references 1, 6, 25, and 37). Of the spliceosomal snRNAs, only U2, U4, and U6 homologs have been identified in trypanosomes (35,49,50). From extensive phylogenetic sequence comparisons, it is evident that many sequence elements and parts of the proposed secondary structures of U2, U4, and U6, such as the U4-U6 base-pairing interaction, are conserved between cis-and trans-spliceosomal systems (18). These common elements may reflect pathways of snRNA maturation, snRNP assembly, and snRNP transport or mechanistic principles that are shared by cis-and transsplicing systems. However, phylogenetic analysis has also revealed that trypanosomal snRNAs principally deviate from their counterparts of other eucaryotes functioning in cis splicing. It will be important to relate these differences in RNA sequence and secondary structure to the organization of the RNA-protein complexes and ultimately to functional, mechanistic differences between cis and trans splicing.In trypanosomes, no homologs of the cis-spliceosomal Ul and U5 snRNPs are known; the SL RNA, in the form of a ribonucleoprotein, may perform the function of the Ul snRNP, catalyzing trans splicing of its own 5'-terminal spliced leader portion (9). In contrast to the trans-spliceosomal U2 and U4/U6 snRNPs, detailed structural information is available on the cis-spliceo...

show abstract

Sedimentation Analysis of Ribonucleoprotein Complexes

Medenbach

Damianov

Schreiner

et al. 2005

Handbook of RNA Biochemistry

View full text Add to dashboard Cite

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

Cited by 53 publications

References 34 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

Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.

Sedimentation Analysis of Ribonucleoprotein Complexes

Contact Info

Product

Resources

About