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PubMed2005

Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed sequence tag database represents sequences derived from rotifers. In summary, the description of SL-mediated trans-splicing in Rotifera extends its representation to at least five metazoan phyla, making it increasingly probable that this is a phylogenetically widespread and therefore ancient phenomenon.

Animals↗

PubMed2003

The Leishmania tarentolae spliced leader contains determinants for association with polysomes.

In kinetoplastids, every nuclear-derived mRNA contains an identical 39-nucleotide (nt) spliced leader at its 5'-terminus. The spliced leader is derived from substrate spliced leader RNA and joined to pre-mRNA by trans-splicing, thus providing mature mRNAs with an m7G cap and additional methylations referred to as cap 4. It was shown previously that mutations spanning nucleotides 10-39 of the spliced leader did not affect substrate spliced leader RNA transcription or trans-splicing in Leishmania tarentolae (Saito, R. M., Elgort, M. G., and Campbell, D. A. (1994) EMBO J. 13, 5460-5469). In this study we examined these sequences for a possible role in translation by assaying the association of mRNAs, which possess mutated spliced leaders, with polysomes. For the nt 28-39 mutated spliced leaders, both the substrate spliced leader RNA and the spliced leader demonstrated a wild-type methylation pattern; spliced nt 28-39 mRNA was found in polysomes. Thus, the nt 28-39 region conserved primary sequence is not a determinant of polysome association. An undermethylated cap 4 structure was present on substrate and mRNA spliced leaders in nt 20-29 mutated exons; nt 20-29 mRNA was not present in polysomes. A differential pattern of cap 4 methylation was seen between the nt 10-19 substrate spliced leader RNA and the nt 10-19 spliced leaders found in the poly(A)+ population of RNA; the nt 10-19 mRNA was not seen in polysomes. Undermethylated spliced leaders did not associate efficiently with polysomes, suggesting a requirement for the cap 4 and/or primary sequence of the spliced leader in translation. This is the first report demonstrating that the spliced leader contains critical structural or sequence determinants for association with polysomes and, hence, translation.

5' Untranslated Regions↗

PubMed1990

trans-spliced Caenorhabditis elegans mRNAs retain trimethylguanosine caps.

The nematode Caenorhabditis elegans has an unusual small nuclear RNA, containing a 100-nucleotide RNA molecule, spliced leader RNA, which donates its 5' 22 nucleotides to a variety of recipient RNAs by a trans-splicing reaction. The spliced leader RNA has a 5' trimethylguanosine (TMG) cap, which becomes the 5' end of trans-spliced mRNAs. We found that mature trans-spliced mRNAs were immunoprecipitable with anti-TMG cap antibodies and that TMG-containing dinucleotides specifically competed with the trans-spliced mRNAs for antibody binding. We also found that these mRNAs retained their TMG caps throughout development and that the TMG-capped mRNAs were polysome associated. Since the large majority of C. elegans mRNAs are not trans-spliced, the addition of the spliced leader and its TMG cap to a limited group of recipient RNAs may create a functionally distinct subset of mRNAs.

Actins↗

PubMed2006

A non-universal transcription factor? The Leishmania tarentolae TATA box-binding protein LtTBP associates with a subset of promoters.

In kinetoplastids a 39-nucleotide spliced leader RNA is trans-spliced to the 5' end of nuclear mRNAs before they can be translated, thus the spliced leader is central to gene expression in kinetoplastid biology. The spliced leader RNA genes in Leishmania tarentolae contain promoters with important sites at approximately -60 and -30. A complex forms specifically on the -60 element as shown by electrophoretic mobility shift. The -60 shift complex has an estimated mass of 159 kDa. An L. tarentolae homologue of TATA-binding protein, LtTBP, co-fractionates with the -60 shift complex. Inclusion of anti-LtTBP antiserum in the shift assay disrupts the shift, indicating that LtTBP is a component of the complex that interacts with the TATA-less -60 element of the spliced leader RNA gene promoter. Both LtTBP and LtSNAP50 are found near the spliced leader RNA gene promoter and the promoters important for tRNAAla and/or U2 snRNA gene transcription, as demonstrated by chromatin immunoprecipitation. The LtTBP appears to interact with a subset of promoters in kinetoplastids with an affinity for short transcription units.

Animals↗

PubMed1994

RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs.

We have identified a new spliced leader (SL) in Fasciola hepatica by characterizing the 5'-terminal sequences of its enolase mRNA, an mRNA also trans-spliced in the flatworm Schistosoma mansoni (Rajkovic, A., Davis, R.E., Simonsen, J.N., and Rottman, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8879-8883). This 37-nucleotide spliced leader is at the 5' ends of multiple Fasciola mRNAs and is likely to be derived from the 5' terminus of a nonpolyadenylated, 108-nucleotide RNA with a trimethylguanosine cap. The SL RNA gene is present in approximately 100 copies within a 1.1-kilobase genomic tandem repeat. Secondary structure predictions indicate that the Fasciola SL RNA contains three stem loops in contrast to two previously observed in S. mansoni. Fasciola and S. mansoni SLs are likely to be evolutionarily related although their sequence identity is only 65%. In contrast with nematodes, absolute conservation of SL sequences and secondary structure does not occur in trematodes. A spliced leader in Fasciola indicates that trans-splicing is likely to be a common feature in other trematodes and perhaps other flatworms.

Amino Acid Sequence↗

PubMed2005

Novel and essential subunits in the 300-kilodalton nuclear cap binding complex of Trypanosoma brucei.

One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m7Gpppm2(6)AmpAmpCmpm3Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-alpha and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing.

Amino Acid Sequence↗

PubMed2002

The spliced leader-associated RNA is a trypanosome-specific sn(o) RNA that has the potential to guide pseudouridine formation on the SL RNA.

The spliced leader-associated (SLA1) RNA is a trypanosome-specific small RNA with unknown function. SLA1 carries a Sm-like site, and is associated with core Sm proteins. Here we found that SLA1 belongs to a family of hairpin-containing RNAs that are implicated in directing pseudouridylation. A potential for base-pair interaction between SLA1 and spliced leader (SL) RNA agrees with the canonical rules for guiding pseudouridylation on SL RNA. Direct RNA analysis showed that this uridine is indeed pseudouridylated in the SL RNA of Leptomonas collosoma, Leishmania major, and Trypanosoma brucei. This position is conserved in all trypanosomatid SL RNAs. Mutations introduced in the SL RNA to disrupt the interaction domain of SLA1/SL RNA abolished the formation of the pseudouridine. SLA1 is localized both to the nucleolus and nucleoplasm. This study solves a long-standing question regarding the function of this novel RNA and describes the first H/ACA RNA, which, unlike all other pseudouridine guides, is also a bona fide snRNA.

Animals↗

PubMedDate not supplied

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae).

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.

Animals↗

PubMed2004

Chemical synthesis and binding activity of the trypanosomatid cap-4 structure.

Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5' end of all mRNAs, thus donating a 5' cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5'-5'-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5' cap includes 2'-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7Gpppm3(6,6,2')Apm2'Apm2' Cpm2(3,2')U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5' terminal mRNA tetranucleotide fragment (pm3(6,6,2')Apm2'Apm2'Cpm2(3,2')U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5'-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.

Animals↗

PubMed1992

Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells.

Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms. Trans-splicing involves an interaction between a 5' splice site present in a spliced leader RNA and a 3' splice site located near the 5' end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3' splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.

Actins↗

PubMed1986

The trypanosome spliced leader small RNA gene family: stage-specific modification of one of several similar dispersed genes.

Diverse mRNAs of Trypanosoma brucei possess the same 5' terminal 35 nucleotides, termed the spliced leader (SL), which appears to be derived from a separate 135 nucleotide transcript. This small SL RNA is encoded within a 1.4 kb unit of DNA which is tandemly reiterated in the genome. In addition, there are at least 4 orphon elements containing SL sequences dispersed from the tandem array. Here we show that during the trypanosome life cycle one of the SL orphons undergoes a stage-specific modification that prevents cleavage of an EcoRV site and we further demonstrate that although only one orphon is modified, three of the SL orphons are flanked by very similar sequences. Each of these contains SL reiteration units including the non-transcribed spacer DNA, suggesting that they did not originate through an RNA intermediate. In addition no evidence of direct repeats at the junction of 1.4 kb and non-1.4 kb DNA was observed. Finally, a phylogenetic survey indicates that while many trypanosomatid species possess similarly organized SL-like sequences, only the SL orphons of closely related subspecies of the T. brucei - T. evansi complex share similar flanking regions.

Animals↗

PubMed1986

The U2 RNA analogue of Trypanosoma brucei gambiense: implications for a splicing mechanism in trypanosomes.

We have isolated the gene coding for the U2 analogue in trypanosomes. The 148 nucleotide long U2 RNA is capped and transcribed from a single copy gene. The 5' half of the molecule is highly homologous to mammalian U2 RNA, while the 3' half does not show any significant sequence homology with the mammalian counterpart. Nevertheless, the trypanosome U2 RNA can be folded into a secondary structure resembling the one proposed for U2 RNA. The presence of a U2 analogue and most likely other U RNAs in trypanosomes suggests that splicing is involved at some point in the maturation of mRNA. Possible interactions of the U2 RNA with the spliced leader RNA are considered.

Animals↗

PubMed1990

mRNAs that mature through trans-splicing in Caenorhabditis elegans have a trimethylguanosine cap at their 5' termini.

Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through a trans-splicing mechanism that involves the transfer of a 22-nucleotide spliced leader to the 5' end of the pre-mRNA. The spliced leader RNA exists as a small nuclear ribonucleoprotein particle and has the trimethylguanosine cap that is characteristic of eucaryotic small nuclear RNAs. We found that the trimethylguanosine cap present on the spliced leader RNA was transferred to the pre-mRNA during the trans-splicing reaction. Thereafter, the trimethylguanosine cap was maintained on the mature mRNA. This is the first example of eucaryotic cellular mRNAs possessing a trimethylguanosine cap structure.

Animals↗

PubMed1994

Kinetics of an RNA conformational switch.

The spliced leader RNA from Leptomonas collosoma has two competing secondary structures of nearly equal free energy. Short, complementary oligonucleotides can drive the structure from one form of the other. We report stopped-flow rapid-mixing and temperature-jump measurements of the kinetics of the structural switch. At high concentrations of oligonucleotide, the rate of binding becomes limited by the rate of the structural switch, which occurs on a time scale of a fraction of a second. The low activation energy observed for the process implies a branch migration type of mechanism in which portions of the two competing helices transiently coexist.

Animals↗

PubMed1998

In vivo function of mutated spliced leader RNAs in Caenorhabditis elegans.

The role of spliced leader RNA (SL RNA) in trans-splicing in Caenorhabditis elegans has been studied through a combination of in vitro mutagenesis and in vivo complementation of rrs-1 mutant nematodes, which lack endogenous SL1 RNA. Three classes of mutant SL1 RNAs have been found-those that rescue the lethal phenotype at low concentration of transforming DNA, those that rescue at high but not low concentration, and those that do not rescue at all. These studies showed that some mutations in the otherwise highly conserved 22-nt spliced leader are tolerated for splicing and post-splicing events. A longer spliced leader also can be tolerated but only when present in high copy number. Changes in the first 16 nucleotides result in the appearance of no SL RNA, consistent with the in vitro studies by others showing that the SL1 RNA promoter partly resides within the spliced leader sequence.

Animals↗

PubMed2000

Tandemly repeated DNA is a target for the partial replacement of thymine by beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

In the DNA of African trypanosomes a small fraction of thymine is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J). The function of this large base is unknown. The presence of J in the silent variant surface glycoprotein gene expression sites and the lack of J in the transcribed expression site indicates that DNA modification might play a role in control of gene repression. However, the abundance of J in the long telomeric repeat tracts and in subtelomeric arrays of simple repeats suggests that J may also have specific functions in repetitive DNA. We have now analyzed chromosome-internal repetitive sequences in the genome of Trypanosoma brucei and found J in the minichromosomal 177-bp repeats, in the long arrays of 5S RNA gene repeats, and in the spliced-leader RNA gene repeats. No J was found in the rDNA locus or in dispersed repetitive transposon-like elements. Remarkably, the rDNA of T. brucei is not organized in long arrays of tandem repeats, as in many other eukaryotes. T. brucei contains only approximately 15-20 rDNA repeat units that are divided over six to seven chromosomes. Our results show that J is present in many tandemly repeated sequences, either at a telomere or chromosome internal. The presence of J might help to stabilize the long arrays of repeats in the genome.

Animals↗

PubMed1999

Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins.

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.

Amino Acid Sequence↗

PubMed1994

Spliced leader-associated RNA of trypanosomes. Sequence conservation and association with protein components common to trans-spliceosomal ribonucleoproteins.

A spliced leader-associated (SLA) RNA recently identified in Trypanosoma brucei has been proposed as a U5 RNA analog in trans-splicing, based on in vivo psoralen cross-linking to the spliced leader (SL) RNA and a short stretch of homology to a conserved U5 RNA sequence. Here we show that the SLA RNA is in the form of an RNP containing common proteins that also occur in the known trans-spliceosomal snRNPs of T. brucei, the SL, U2, and U4/U6 snRNPs. Therefore the SLA RNP is a member of a larger family of related RNPs. The immunoprecipitation of the SLA RNA by antibodies against common proteins does not require an association between the SLA RNA and the SL RNP. To identify sequences of potential functional importance and to establish a secondary structure model, we have sequenced the SLA RNA and cloned its gene from a related trypanosomatid, Leptomonas seymouri. A sequence comparison between the T. brucei and L. seymouri SLA RNAs reveals that the U5-homologous sequence element is not conserved.

Animals↗