Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

Patzelt, E; Perry, Karen L; Agabian, Nina

doi:10.1128/mcb.9.10.4291

Cited by 44 publications

(34 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ␣-tubulin UR also contains an AC dinucleotide preceding the AG at the 3ЈSS. One possible explanation for our observation was that the previous 3ЈSS assignment was incorrect (32). However, we discarded this hypothesis after sequencing of ␣-tubulin cDNA clones confirmed the original assignment, which was also verified by primer extension (see below).…”

Section: Resultsmentioning

confidence: 94%

“…In contrast to cis splicing, trans splicing joins exons derived from two independently transcribed RNAs. trans splicing and cis splicing, however, share remarkable similarities: both require the same characteristic sequence motifs [GT at the 5ЈSS, an adenosine branch point, a poly(Y) tract, AG at the 3ЈSS, and possibly exonic enhancer motifs] (15,16,23,27,30,32,40), both follow the same general mechanism (two catalytic trans esterification reactions), and many of the major components of the yeast or human spliceosome are conserved in T. brucei (22). trans splicing is a prerequisite for protein expression, and changes in the poly(Y) tract, leading to a difference in trans-splicing efficiency, should be reflected in differences in protein levels.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

Siegel

Tan

Cross

2005

Molecular and Cellular Biology

View full text Add to dashboard Cite

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5 splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3 splice sites (3SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3SS, we constructed a luciferase-␤-galactosidase doublereporter system. By testing ϳ90 sequences, we demonstrated that the optimum poly(Y) tract length is ϳ25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased transsplicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3SS is important, and an AC dinucleotide at positions ؊3 and ؊4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.In eukaryotes, a central step in generating mature mRNA from pre-mRNA is the removal of introns and the joining of the two flanking exons, a process known as cis splicing (14, 33). For successful splicing, the intron has to be correctly identified. Several sequence elements are implicated in defining the two splice sites. The 5Ј end of the intron is generally defined by a GT dinucleotide, whereas the 3Ј end is marked by AG. Additional characteristics of the 3Ј splice site (3ЈSS) are a branch point sequence followed by a pyrimidine-rich [poly(Y)] tract. Enhancer regions, which may contribute to the assembly of the spliceosome or the identification of the correct 3ЈSS, have also been described (33). Splicing involves two trans esterification steps. During the first trans esterification, the conserved branch point adenosine forms a 2Ј-to-5Ј phosphodiester bond with the 5Ј end of the intron (13, 34). This step depends on U2 snRNP binding to the branch point sequence, which in turn requires the help of the heterodimeric auxiliary factor U2AF, consisting of 65-kDa and 35-kDa subunits. The U2AF65 subunit has been shown to bind to the poly(Y) tract, whereas U2AF35 associates with the 3ЈSS AG dinucleotide (20,46,47). During the second trans esterification, the free hydroxyl of the upstream exon attacks the phosphate of the 3ЈSS to join the exons and release the lariat-shaped intron (25).The mechanism for selecting the appropriate 3ЈSS AG dinucleotide from other cryptic AG sites for the second trans esterification reaction has not entirely been solved. Two models have been proposed. According to the scanning model, identification of the correct 3...

show abstract

Section: Resultsmentioning

confidence: 94%

mentioning

confidence: 99%

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

Siegel

Tan

Cross

2005

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…The reaction was analyzed on a 6% denaturing polyacrylamide gel next to a DNA sequencing reaction with the same primer. Primer extension sequencing of the rRNA was carried out as previously described (41).…”

Section: Methodsmentioning

confidence: 99%

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

Xu¹,

Liu²,

López-Estraño³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

We analyzed three chromosomal loci of the trypanosomatid Leptomonas collosoma encoding box C/D small nucleolar RNAs (snoRNAs). All the snoRNAs that were analyzed here carry two sequences complementary to rRNA target sites and obey the ؉5 rule for guide methylation. Studies on transgenic parasites carrying the snoRNA-2 gene in the episomal expression vector (pXneo) indicated that no promoter activity was found immediately adjacent to this gene. Deleting the flanking sequences of snoRNA-2 affected the expression; in the absence of the 3-flanking (but not 5-flanking) sequence, the expression was almost completely abolished. The snoRNA genes are transcribed as polycistronic RNA. All snoRNAs can be folded into a common stem-loop structure, which may play a role in processing the polycistronic transcript. snoRNA B2, a member of a snoRNA cluster, was expressed when cloned into the episomal vector, suggesting that each gene within a cluster is individually processed. Studies with permeable cells indicated that snoRNA gene transcription was relatively sensitive to ␣-amanitin, thus supporting transcription by RNA polymerase II. We propose that snoRNA gene expression, similar to protein-coding genes in this family, is regulated at the processing level.

show abstract

“…The alkaline partial hydrolysis ladder was produced by boiling the RNA in 50 mM NaOH and 1 mM EDTA for 2 min. Primer extension was as described (9,18). Northern Blot Analysis.…”

Section: Methodsmentioning

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

Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

Cited by 44 publications

References 30 publications

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

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

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

Contact Info

Product

Resources

About