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PubMed1999

Trypanosoma cruzi: suppression of tuzin gene expression by its 5'-UTR and spliced leader addition site.

Teixeira, S. M. R., Kirchhoff, L. V., and Donelson, J. E. 1999. Trypanosoma cruzi: Suppression of tuzin gene expression by its 5'-UTR and spliced leader addition site. Experimental Parasitology 93, 143-151. The genome of the protozoan parasite Trypanosoma cruzi contains a tandemly repeated array of two alternating genes, one encoding amastin and the other encoding tuzin. Amastin is an abundant amastigote surface protein, whereas tuzin is thought to be a rare protein whose location and function are unknown. The 137-nucleotide 5' untranslated region (5'-UTR) of the tuzin mRNA has a 22-codon open translation reading frame containing 3 methionine codons followed by a stop codon that overlaps the methionine start codon of the tuzin coding region. A fragment containing the tuzin 5'-UTR and upstream intergenic region was placed in front of a luciferase reporter gene in a plasmid for transient transfection assays of luciferase activity. By mutating the three upstream ATGs in the tuzin 5'-UTR and replacing the tuzin spliced leader (SL) acceptor site with that of the amastin gene, we found that the 22-codon reading frame and the tuzin SL acceptor site combine to substantially reduce expression of the luciferase gene. These results indicate that expression of the multicopy tuzin gene is posttranscriptionally suppressed by both inefficient RNA processing and poor translation initiation, resulting in a low level of tuzin.

5' Untranslated Regions↗
PubMed1993

Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing.

To probe functions of the U1 small nuclear ribonucleoprotein particle (snRNP) during in vitro splicing, we have used unusual splicing substrates which replace the 5' splice site region of an adenovirus substrate with spliced leader (SL) RNA sequences from Leptomonas collosoma or Caenorhabditis elegans. In agreement with previous results (J.P. Bruzik and J.A. Steitz, Cell 62:889-899, 1990), we find that oligonucleotide-targeted RNase H destruction of the 5' end of U1 snRNA inhibits the splicing of a standard adenovirus splicing substrate but not of the SL RNA-containing substrates. However, use of an antisense 2'-O-methyl oligoribonucleotide that disrupts the first stem of U1 snRNA as well as stably sequestering positions of U1 snRNA involved in 5' and 3' splice site recognition inhibits the splicing of both the SL constructs and the standard adenovirus substrate. The 2'-O-methyl oligoribonucleotide is no more effective than RNase H pretreatment in preventing pairing of U1 with the 5' splice site, as assessed by inhibition of psoralen cross-link formation between the SL RNA-containing substrate and U1. The 2'-O-methyl oligoribonucleotide does not alter the protein composition of the U1 monoparticle or deplete the system of essential splicing factors. Native gel analysis indicates that the 2'-O-methyl oligoribonucleotide inhibits splicing by diminishing the formation of splicing complexes. One interpretation of these results is that removal of the 5' end of U1 inhibits base pairing in a different way than sequestering the same sequence with a complementary oligoribonucleotide. Alternatively, our data may indicate that two elements near the 5' end of U1 RNA normally act during spliceosome assembly; the extreme 5' end base pairs with the 5' splice site, while the sequence or structural integrity of stem I is essential for some additional function. It follows that different introns may differ in their use of the repertoire of U1 snRNP functions.

Adenoviridae↗
PubMed1990

Permeable trypanosome cells as a model system for transcription and trans-splicing.

We have established conditions for Trypanosoma brucei permeable cells to study transcription and trans-splicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCl, whereas higher potassium concentrations were inhibitory. In addition, MgCl2 concentrations above 3 mM led to the accumulation of a 3' end shortened SL RNA species, which has been previously reported not to participate in trans-splicing. Using conditions optimal for the synthesis of the SL RNA, we observed accurate trans-splicing of newly-synthesized alpha-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced alpha-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.

Amanitins↗
PubMed2004

A survey of SL1-spliced transcripts from the root-lesion nematode Pratylenchus penetrans.

Plant-parasitic nematodes are important and cosmopolitan pathogens of crops. Here, we describe the generation and analysis of 1928 expressed sequence tags (ESTs) of a splice-leader 1 (SL1) library from mixed life stages of the root-lesion nematode Pratylenchus penetrans. The ESTs were grouped into 420 clusters and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. Approximately 80% of all translated clusters show homology to Caenorhabditis elegans proteins, and 37% of the C. elegans gene homologs had confirmed phenotypes as assessed by RNA interference tests. Use of an SL1-PCR approach, while ensuring the cloning of the 5' ends of mRNAs, has demonstrated bias toward short transcripts. Putative nematode-specific and Pratylenchus -specific genes were identified, and their implications for nematode control strategies are discussed.

Animals↗
PubMed2004

Contribution of trans-splicing, 5' -leader length, cap-poly(A) synergism, and initiation factors to nematode translation in an Ascaris suum embryo cell-free system.

Trans-splicing introduces a common 5' 22-nucleotide sequence with an N-2,2,7-trimethylguanosine cap (m (2,2,7)(3)GpppG or TMG-cap) to more than 70% of transcripts in the nematodes Caenorhabditis elegans and Ascaris suum. Using an Ascaris embryo cell-free translation system, we found that the TMG-cap and spliced leader sequence synergistically collaborate to promote efficient translation, whereas addition of either a TMG-cap or spliced leader sequence alone decreased reporter activity. We cloned an A. suum embryo eIF4E homolog and demonstrate that this recombinant protein can bind m(7)G- and TMG-capped mRNAs in cross-linking assays and that binding is enhanced by eIF4G. Both the cap structure and the spliced leader (SL) sequence affect levels of A. suum eIF4E cross-linking to mRNA. Furthermore, the differential binding of eIF4E to a TMG-cap and to trans-spliced and non-trans-spliced RNAs is commensurate with the translational activity of reporter RNAs observed in the cell-free extract. Together, these binding data and translation assays with competitor cap analogs suggest that A. suum eIF4E-3 activity may be sufficient to mediate translation of both trans-spliced and non-trans-spliced mRNAs. Bioinformatic analyses demonstrate the SL sequence tends to trans-splice close to the start codon in a diversity of nematodes. This evolutionary conservation is functionally reflected in the optimal SL to AUG distance for reporter mRNA translation in the cell-free system. Therefore, trans-splicing of the SL1 leader sequence may serve at least two functions in nematodes, generation of an optimal 5'-untranslated region length and a specific sequence context (SL1) for optimal translation of trimethylguanosine capped transcripts.

5' Untranslated Regions↗
PubMed2003

Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display.

All parasitic helminths process a large subset of their mRNA molecules via spliced leader trans-splicing. Here we describe a rapid and efficient method, 'spliced leader differential display', to generate comprehensive expression fingerprints of trans-spliced factors on silver-stained acrylamide gels. The method allows systematic screening of the entire trans-spliced transcriptome of parasitic organisms using only minute amounts of starting material. Spliced leader differential display selectively displays 5' end-fragments of trans-spliced messages thus facilitating the identification of associated proteins and the full-length characterisation of corresponding mRNAs. We employed spliced leader differential display to analyse gene expression patterns of in vitro cultivated protoscoleces and metacestode vesicles from Echinococcus multilocularis. These analyses revealed that the majority of trans-spliced mRNAs are expressed at equal amounts in both larval stages whereas about 25% of the identified transcripts displayed stage-specific differential expression. Full-length transcripts for several of the obtained fragments were isolated and their constitutive or differential expression was verified by competitive PCR and Virtual Northern blotting. Spliced leader differential display was further used to identify genes which are induced in the E. multilocularis metacestode stage under growth promoting conditions in the presence of host hepatocytes. One mRNA, encoding a putative member of the epidermal growth factor family of mitogens, was shown to be 10-fold induced upon co-incubation of metacestode vesicles with host cells which indicates a possible involvement of parasite-determined, epidermal growth factor-like signal transduction systems in metacestode proliferation and development. The successful application of spliced leader differential display to gene expression analyses of echinococcal larvae, combined with easy handling and a low rate of false positives, recommends this method not only for studies on the host-parasite interplay during alveolar echinococcosis, but also for investigations on other parasitic organisms.

Adolescent↗
PubMed2000

Structure and sequence variation of the trypanosome spliced leader transcript.

We have assessed the potential of using the spliced leader (SL) or mini-exon gene as a marker for molecular phylogenetic analysis of genus Trypanosoma. A total of 27 trypanosome sequences were compared, 18 of these being newly reported. In contrast to genus Leishmania, we found the non-transcribed spacer region of the SL locus in trypanosomes to be far too variable for informative comparison of all but the most closely related species. At the other extreme, the short (39 nt) SL exon was usually completely conserved and hence uninformative. The SL RNA showed variation in both length (97-152 nt) and sequence among different trypanosome species, with most variation occurring in stem-loop II. Consequently, this region could not be aligned with confidence in multiple sequence alignment, severely reducing the number of phylogenetically informative nucleotide positions. In computer simulation, most of the SL RNAs readily folded into the 3 stem-loop secondary structure predicted previously, but again stem-loop II was highly variable. No obvious correlation could be seen between the length of this stem-loop and trypanosome biology. We conclude that the SL repeat is not an informative phylogenetic marker for long range evolutionary studies of genus Trypanosoma.

Animals↗
PubMed2004

Nematode m7GpppG and m3(2,2,7)GpppG decapping: activities in Ascaris embryos and characterization of C. elegans scavenger DcpS.

A spliced leader contributes the mature 5'ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m3(2,2,7)GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3' to 5' pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is approximately 15-fold less active than the 3' to 5' pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m3(2,2,7)GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5' ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m3(2,2,7)GpppG.

Animals↗
PubMed2004

Efficient replication of full-length murine leukemia viruses modified at the dimer initiation site regions.

Retroviruses encapsidate two copies of full-length viral RNA molecules linked together as a dimeric genome. RNA stem loop structures harboring palindromic (or "kissing") loop sequences constitute important cis-elements for viral dimerization known as dimer initiation sites (DIS). In murine leukemia virus (MLV), a 10-mer and a 16-mer palindrome (DIS-1 and DIS-2, respectively) located in the viral leader region mediate dimerization in vitro and affect dimer stability of vector RNA in vivo. We have investigated the effect on viral replication of introducing deletions or nucleotide substitutions within these palindromes in a full-length MLV genome. Our results demonstrate that viruses modified at the dimer initiation site regions are viable and show wild-type levels of RNA encapsidation. One mutant lacking the DIS-1 palindrome was severely impaired and displayed an increased cellular ratio of spliced versus genomic RNA that most likely contributes to the inefficient replication. The implications for development of DIS-modified retrovirus-based vectors are discussed.

5' Untranslated Regions↗
PubMedDate not supplied

A PCR-based survey on Phytomonas (Euglenozoa: Trypanosomatidae) in phytophagous hemipterans of the Amazon region.

We have surveyed 244 hemipterans from Western Brazilian Amazĵnia for the presence of trypanosomatids and identification of members of the genus Phytomonas. Examination by phase microscopy of squashes of insect salivary glands (SG) and digestive tubes (DT) revealed that 44% (108/244) of insects from seven families harbored trypanosomatids. Infections were 5 times more frequent in Coreidae than in all other families together. Smears of SG and DT of the dissected insects were fixed on glass slides with methanol and stained with Giemsa for morphological analysis. DNA was recovered from these preparations and submitted to a PCR assay that permitted amplification of all trypanosomatid genera using primers of conserved sequences flanking a segment of the spliced leader (SL) gene. Upon PCR amplification of the recovered DNA, amplicons were hybridized with an oligonucletide probe (SL3') complementary to a SL intron sequence specific for flagellates of the genus Phytomonas. Among the trypanosomatid-positive insects, 38.8% harbored Phytomonas spp., corresponding to an overall Phytomonas prevalence of 17.1% among phytophagous bugs, their putative vectors. Since many Phytomonas are pathogenic in plants, this high prevalence in their vectors emphasizes the permanent risk of exposure to disease by native and cultured plants of the Amazon region.

Animals↗
PubMed1999

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears.

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.

Animals↗
PubMed2005

Ploidy changes associated with disruption of two adjacent genes on Leishmania major chromosome 1.

Leishmania major Friedlin (LmjF) is a kinetoplastid protozoan whose genomic sequence has been recently elucidated. About 60% of the identified genes do not have a known function, and many are trypanosomatid-specific. Here we characterise two adjacent genes from LmjF chromosome 1 (chr1): LmjF01.0750, which encodes a predicted protein with a serine/threonine protein kinase motif and LmjF01.0760, which encodes a product with no similarity to other known proteins. Orthologues of both genes are present in Trypanosoma cruzi, but neither occur in Trypanosoma brucei. We have mapped polyadenylation and spliced-leader acceptor sites for both genes, and show that they differ between Leishmania species. Attempts to generate null mutants of LmjF01.0750 by homologous recombination were unsuccessful and led to the apparent triploidy of the entire genome, suggesting that it is an essential gene. Interestingly, at least two copies of LmjF01.0750 are required for cell survival. Further evidence of genome plasticity in Leishmania was provided by changes in chr1 copy number that occurred during in vitro growth of wild-type LmjF promastigotes and following replacement of a single copy of LmjF01.0760.

Animals↗
PubMed2001

Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence.

The sequence of the spliced-leader gene repeat of a Brazilian Trypanosoma vivax stock from cattle showed high similarity to sequences of West African T. vivax in both intron and intergenic sequences. This is the first evidence based on DNA sequences of close-relatedness between Brazilian and West African T. vivax stocks. A T. vivax-specific diagnostic PCR assay based on spliced-leader gene intergenic sequences was able to amplify DNA from T. vivax stocks from South America (Brazil, Bolivia, and Colombia) and West Africa. Species-specificity of this method was confirmed by results obtained by testing 15 other trypanosomes, including other species and subspecies that can also infect cattle. The PCR assay developed presented high sensitivity, detecting the DNA content of only one parasite and also revealing T. vivax infection in asymptomatic animals without detectable parasitemia by microhematocrit or in Giemsa-stained blood smears. Use of crude preparations from field-blood samples collected on both filter paper and glass slides as DNA template suggested that this method could be useful for the diagnosis of T. vivax in large epidemiological studies.

Animals↗
PubMed1999

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene.

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.

Animals↗
PubMed2005

Dehydration-induced tps gene transcripts from an anhydrobiotic nematode contain novel spliced leaders and encode atypical GT-20 family proteins.

Accumulation of the non-reducing disaccharide trehalose is associated with desiccation tolerance during anhydrobiosis in a number of invertebrates, but there is little information on trehalose biosynthetic genes in these organisms. We have identified two trehalose-6-phosphate synthase (tps) genes in the anhydrobiotic nematode Aphelenchus avenae and determined full length cDNA sequences for both; for comparison, full length tps cDNAs from the model nematode, Caenorhabditis elegans, have also been obtained. The A. avenae genes encode very similar proteins containing the catalytic domain characteristic of the GT-20 family of glycosyltransferases and are most similar to tps-2 of C. elegans; no evidence was found for a gene in A. avenae corresponding to Ce-tps-1. Analysis of A. avenae tps cDNAs revealed several features of interest, including alternative trans-splicing of spliced leader sequences in Aav-tps-1, and four different, novel SL1-related trans-spliced leaders, which were different to the canonical SL1 sequence found in all other nematodes studied. The latter observation suggests that A. avenae does not comply with the strict evolutionary conservation of SL1 sequences observed in other species. Unusual features were also noted in predicted nematode TPS proteins, which distinguish them from homologues in other higher eukaryotes (plants and insects) and in micro-organisms. Phylogenetic analysis confirmed their membership of the GT-20 glycosyltransferase family, but indicated an accelerated rate of molecular evolution. Furthermore, nematode TPS proteins possess N- and C-terminal domains, which are unrelated to those of other eukaryotes: nematode C-terminal domains, for example, do not contain trehalose-6-phosphate phosphatase-like sequences, as seen in plant and insect homologues. During onset of anhydrobiosis, both tps genes in A. avenae are upregulated, but exposure to cold or increased osmolarity also results in gene induction, although to a lesser extent. Trehalose seems likely therefore to play a role in a number of stress responses in nematodes.

Amino Acid Sequence↗
PubMed2003

Operon structure and trans-splicing in the nematode Pristionchus pacificus.

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.

Amino Acid Sequence↗
PubMed2005

Mitochondrial leader sequence--plasmid DNA conjugates delivered into mammalian cells by DQAsomes co-localize with mitochondria.

In the last decade the increase in therapeutic strategies aimed at mitochondrial targets has resulted in the need for novel delivery systems for the selective delivery of drugs and DNA into mitochondria. In this study, we have continued our efforts towards the development of the first mitochondriotropic drug and DNA delivery system (DQAsomes). Prepared from derivatives of the self-assembling mitochondriotropic bola-amphiphile dequalinium chloride, these vesicles bind and transport DNA to mitochondria in living mammalian cells where upon they have been shown to release the DNA on contact with mitochondrial membranes. We present data to demonstrate that oligonucleotides as well as plasmid DNA conjugated to a mitochondrial leader sequence (MLS) co-localize with mitochondria when delivered into mammalian cells by DQAsomes. In contrast to a commercially available DNA delivery vector, our vesicles appear to have a pronounced specificity for mitochondria. Further, the data strongly suggest that linear conjugates might be better suited to delivery into mitochondria and that in the absence of a mitochondria specific vector, the presence of a MLS-peptide conjugated to the DNA is alone not sufficient to direct the accumulation of DNA at mitochondria.

Cell Line, Tumor↗