mRNA splicing in Trypanosoma brucei: Branch-point mapping reveals differences from the canonical U2 snRNA-mediated recognition

Lücke, Stephan; Jürchott, Karsten; Hung, Lee-Hsueh; Bindereif, Albrecht

doi:10.1016/j.molbiopara.2005.04.007

Cited by 14 publications

(18 citation statements)

References 15 publications

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“…Furthermore, our data suggest that changes in length between the branch point and splice site are acceptable as long as the poly(Y) tract and spacer region fulfill certain requirements. Our observations are consistent with recently published data indicating that T. brucei genes do not necessarily contain a single branch point and that different nucleotides may serve as branch points (24). One of the early events in spliceosome assembly is the recruitment of U2 snRNP to the branch point sequence.…”

Section: Role Of Poly(y) Tractsupporting

confidence: 92%

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

Siegel

Tan

Cross

2005

Molecular and Cellular Biology

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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...

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Section: Role Of Poly(y) Tractsupporting

confidence: 92%

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

Siegel

Tan

Cross

2005

Molecular and Cellular Biology

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“…This approach, i.e., PCR amplification across the branch point, was also recently used to identify branch points for trans-spliced Trypanosoma brucei RNAs. Here too, multiple branch points per intron were detected (27). Most were A branch points, but C branch points were observed.…”

Section: Resultsmentioning

confidence: 82%

Branch Point Identification and Sequence Requirements for Intron Splicing in Plasmodium falciparum

et al. 2011

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Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intronexon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5 and 3 splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5 and 3 splice sites. However, the 5 consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3 splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack.Introns are noncoding sequences located inside precursor mRNA (pre-mRNA) transcripts that are excised before nuclear export (39). Splicing of pre-mRNA requires sequence motifs in the intron and is mediated by a ribonucleoprotein complex called the spliceosome (37,53). While the fundamental mechanisms of splicing are conserved among eukaryotes (4), splice site recognition motifs are short and often only weakly conserved between organisms (20). In addition, most predictive software relies on model organisms, and there has been only limited experimental characterization of intron splicing in deep-branching eukaryotic organisms (50). Introns are predicted to be present in approximately half the genes in the Plasmodium falciparum genome (11). However, inaccuracies in intron prediction have been reported (26,32,42). P. falciparum has an extremely AT-rich genome (11), which has implications for both evolutionary adaptations of the spliceosome machinery and accuracy of gene structure predictions.All spliceosomal introns contain 5Ј donor and 3Ј acceptor splice sites, usually with GU and AG dinucleotides at the respective intron ends and a branch point located within the intron. Major-class introns often contain a polypyrimidine tract adjacent to the 3Ј splice site, which is missing in minor-class introns (29,39). During splicing, the branch point nucleotide initiates a nucleophilic attack on the 5Ј donor splice site. The free end of the upstream intron then initiates a second nucleophilic attack on the 3Ј acceptor splice site, releasing the intron as an RNA lariat and covalently combining the two exons (53). Intron size variation is largely due to the distance from the intron's 5Ј bound...

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“…Conserved sequences are provided below the drawing with invariant residues underlined. While in mammalian systems, 5ЈSSs, BPs, and 3ЈSSs exhibit partly conserved sequences (R, purine; Y, pyrimidine; N, any base), there is no obvious sequence conservation at trypanosome BPs (43) and 3ЈSSs, although it was shown for the latter that an AC dinucleotide (*) preceding the AG residues drastically reduces splicing efficiency unless a compensatory AG dinucleotide is present within the 5Ј untranslated region (76). It appears that the importance of the polypyrimidine tract becomes more important when consensus sequences are lacking.…”

Section: U Snrnps and The Spliceosome In Higher Eukaryotesmentioning

confidence: 99%

“…Yeast has highly conserved splice site and BP sequences, and some yeast introns function without a polypyrimidine tract (not shown). The partly conserved sequences in mammals require a small polypyrimidine tract in the range of 10 to 12 residues (Y 10-12 ), whereas in trypanosomes, the polypyrimidine tract is large (Y ϳ20 ), is an essential sequence determinant for efficient splicing, and typically starts just downstream of the BP (43,76). After the first transesterification reaction, cis splicing results in a lariat intron structure, whereas a Y structure intermediate is formed in the SL trans splicing process.…”

Section: U Snrnps and The Spliceosome In Higher Eukaryotesmentioning

confidence: 99%

“…For example, human and yeast U2 snRNAs share a conserved motif which is complementary to the BP sequence. Conversely, in trypanosomes, there is no conserved BP sequence and, typically, no complementarity between BP and U2 snRNA sequences (43). Possibly the U2-specific Sm paralogs undergo specific protein-protein interactions that position the U2 snRNP at the BP in the absence of sequence complementarity.…”

Section: Trypanosome Sm and Lsm Proteins And Sm Core Variation In U2 mentioning

confidence: 99%

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The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?

2010

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Trypanosomatids are early-diverged, protistan parasites of which Trypanosoma brucei, Trypanosoma cruzi, and several species of Leishmania cause severe, often lethal diseases in humans. To better combat these parasites, their molecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL) trans splicing in T. brucei established a key difference between parasites and hosts. In SL trans splicing, the capped 5-terminal region of the small nuclear SL RNA is fused onto the 5 end of each mRNA. This process, in conjunction with polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA by providing the cap structure. The reaction is a two-step transesterification process analogous to intron removal by cis splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to ϳ170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicing factors were identified by standard genome annotation because trypanosomatid amino acid sequences are among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterization of the splicing factor repertoire in T. brucei, achieved by tandem affinity purification of splicing complexes, by systematic analysis of proteins containing RNA recognition motifs, and by mining the genome database. In addition, recent findings about functional differences between trypanosome and human pre-mRNA splicing factors are discussed.Trypanosomatids are protistan parasites infecting hosts as diverse as mammals, insects, and plants. In humans, vectorborne Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. cause lethal diseases, and the strong impact of these parasites on global health has spurred investigations of the molecular processes in these organisms from early on. One of the first key discoveries in regard to gene expression was spliced leader (SL) trans splicing, which was eventually found to be an essential maturation step for all nuclear pre-mRNA in trypanosomatids.The initial discoveries of SL trans splicing were made in T. brucei, and until now, this organism has remained the preferred trypanosomatid organism for spliceosomal studies. T. brucei is an extracellular parasite which evades the mammalian immune system by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG expression has therefore been a research focus, and it was on VSG mRNAs that the 5Ј-terminal region was first discovered to contain a leader sequence which was not encoded in the VSG gene (10, 87). Further analysis showed that the 39-nucleotide (nt)-long leader was derived from the 5Ј terminus of a separate, small nuclear RNA, which has been termed SL RNA or miniexon-derived RNA (12,34,56). The discovery of a Y structure intermediate which corresponds to the cis splicing intron-exo...

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mRNA splicing in Trypanosoma brucei: Branch-point mapping reveals differences from the canonical U2 snRNA-mediated recognition

Cited by 14 publications

References 15 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

Branch Point Identification and Sequence Requirements for Intron Splicing in Plasmodium falciparum

The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?

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