Trans-splice Acceptor Site Research Articles

Improving genome-wide mapping of nucleosomes in Trypanosome cruzi.

In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Cross-validated methods for promoter/transcription start site mapping in SL trans-spliced genes, established using the Ciona intestinalis troponin I gene.

In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5′-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5′-leader trans-splicing (SL trans-splicing) because the original 5′-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5′-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA’s original 5′-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla.

Conserved elements associated with ribosomal genes and their trans-splice acceptor sites in Caenorhabditis elegans

The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression.

Conserved nucleotides surrounding the trans-splicing acceptor site and the translation initiation codon in Leishmania genes

Conserved nucleotides surrounding the trans-splicing acceptor site and the translation initiation codon in Leishmania genes

Cyclophilin and protein disulfide isomerase genes are co-transcribed in a functionally related manner in Caenorhabditis elegans.

The ubiquitous enzymes peptidyl prolyl cis-trans isomerase (PPI, EC 5.2.1.8) and protein disulfide isomerase (PDI, EC 5.3.4.1) are important rate-limiting catalysts of protein-folding events in the cell. In the free-living nematode Caenorhabditis elegans, two genes encoding these enzymes (cyp-9 and pdi-1, respectively) are clustered together on chromosome III. In work described elsewhere, the encoded enzymes have been expressed as recombinant proteins and have been determined to possess in vitro PPI and PDI activity. Taken together, this organization of the two genes and the related functions of their transcripts indicate that they may be cotranscribed as a polycistronic unit, similar to bacterial operons. This study details the very close linkage of pdi-1 and cyp-9, which are in the same orientation. pdi-1 is the upstream gene, and the putative polyadenylation cleavage signal of this gene is separated from the trans-splice acceptor site of cyp-9 by only 103 bp. pdi-1 is trans-spliced by the ubiquitous nematode trans-spliced leader SL1, whereas cyp-9 was found to be predominantly trans-spliced by the "operon-specific" trans-spliced leader SL2. Similar trends in relative transcript abundance were demonstrated with synchronously produced mRNA for both genes during larval development, supporting the contention that the genes are co-expressed. Finally, reporter gene analysis provides strong evidence that both genes are controlled by a single upstream regulatory element, which directs expression of both enzymes in the hypodermal cells that synthesize the cuticle.

Gene linkage and steady state RNAs suggest trans-splicing may be associated with a polycistronic transcript in Schistosoma mansoni

Spliced leader (SL) trans-splicing generates the 5′ end of mature mRNAs through the addition of a small exon to pre-mRNAs in some flagellates (kinetoplastida and euglenoids) and metazoans (nematodes and flatworms). Although SL addition in the kinetoplastida and a subset of nematode genes serves to resolve multicistronic mRNAs into monocistronic, capped mRNAs, information regarding the functional significance of trans-splicing in flatworms is limited. We describe here the identification and characterization of a closely linked gene upstream from the trans-spliced enolase gene in the flatworm Schistosoma mansoni. This gene produces a non- trans-spliced mRNA encoding a ubiquinol binding protein, UbCRBP, that is a component of the ubiquinol-cytochrome C reductase complex. The distance between the UbCRBP polyadenylation site and the enolase trans-splice acceptor site is exceptionally short, only 54 nucleotides. Primer extension (5′ RACE), RT-PCR, and RNase mapping have identified steady state, cis-spliced RNAs which significantly overlap both the UbCRBP and enolase genes. These transcripts contain the 5′ ends of mature UbCRBP mRNAs, extend through UbCRBP, across the intergenic region, and a significant distance 3′ into the enolase gene. Interestingly, the close linkage between the UbCRBP and enolase genes is conserved in a second flatworm, Fasciola hepatica, which also trans-splices the downstream enolase gene. Taken together, the role of SL addition in resolving multicistronic transcripts in both C. elegans and the kinetoplastida, the conservation of UbCRBP/enolase gene linkage in two divergent trematodes, and the multicistronic organization of schistosome UbCRBP/enolase RNAs are consistent with the suggestion that these two genes are likely to be cotranscribed and that trans-splicing in flatworms may be associated with polycistronic transcripts.

A transformation vector for stage-specific expression of heterologous genes in Trypanosoma cruzi epimastigotes.

To express heterologous genes in a stage-specific manner, we constructed a transformation vector for Trypanosoma cruzi containing a selection gene (hyg) and a reporter gene (luc) flanked by sequences of the multicopy 1f8 gene arranged so as to provide a trans-splicing acceptor site to hyg and a putative polyadenylation signal to luc. The intergenic region of the T. cruzi genes 294 and KAP was placed between hyg and luc, contributing the polyadenylation signal of 294 to hyg and the KAP trans-splicing acceptor site to luc. Transformation was carried out by electroporation, and transformed epimastigotes were selected in medium containing hygromycin B. Through double homologous recombination of the 1f8 sequences with their chromosomal counterparts, the construction is inserted into the 1f8 locus, substituting probably one and no more than a few copies of the 1f8 gene without having apparent deleterious effects on the parasite. cDNA analysis demonstrated that the introduced signals were correctly processed, resulting in translatable hyg and luc mRNAs. Whereas epimastigotes express luciferase, no expression is found in the trypomastigote stage.

A putative pyruvate dehydrogenase α subunit gene from Trypanosoma cruzi

A full-length DNA clone encoding a putative pyruvate dehydrogenase α subunit (E1 α) gene was isolated from a Trypanosoma cruzi (RA strain) DNA library. Sequencing of this clone revealed it to encode a 378 amino acid protein ( M r 42774) with high sequence similarity to E1 α obtained from different sources. The highest score is obtained with human E1 α: 43,3% similarity. Southern blot analysis is consistent with the existence of a single copy of this putative T. cruzi E1 α gene per haploid genome in different parasite strains. Expression of this gene was demonstrated by Northern blot analysis and its trans-splicing acceptor site was identified by Polymerase Chain Reaction-mediated amplication of its cDNA.

Transient expression mediated by the Trypanosoma cruzi rRNA promoter

Plasmid constructs containing a putative Trypanosoma cruzi rRNA promoter and transcription start point upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene were transfected into cultured T. cruzi epimastigotes to verify the presence of a promoter activity. Constructs bearing the putative promoter and a 3′ trans-splicing acceptor site in the proper orientation yielded approx. two orders of magnitude greater CAT expression than that previously observed with the T. cruzi spliced leader (SL) gene promoter. In contrast, similar constructs lacking the known 3′ splice site yielded reduced but readily measurable expression suggesting that sequences near the promoter may function as cryptic 3′ splice sites. A repeated sequence upstream from the putative basal rRNA promoter in a position analogous to rRNA gene enhancer elements in other eukaryotes did not enhance expression from the T. cruzi rRNA promoter. Finally, these constructs were functional in some but not all T. cruzi isolates, and were inactive in other kinetoplastid species, suggesting that the T. cruzi rRNA promoter may have a limited host range.

Functional analysis of a C. elegans trans-splice acceptor.

The rol-6 gene is trans-spliced to the 22 nt leader, SL1, 173 nt downstream of the transcription start. We have analyzed splicing in transformants carrying extrachromosomal arrays of rol-6 with mutations in the trans-splice acceptor site. This site is a close match to the consensus, UUUCAG, that is highly conserved in both trans-splice and intron acceptor sites in C. elegans. When the trans-splice site was inactivated by mutating the perfectly-conserved AG, trans-splicing still occurred, but at a cryptic site 20 nt upstream. We tested the frequency with which splicing switched from the normal site to the cryptic site when the pyrimidines at this site were changed to A's. Since most C. elegans 3' splice sites lack an obvious polypyrimidine tract, we hypothesized that these four pyrimidines might play this role, and indeed mutation of these bases caused splicing to switch to the cryptic site. We also demonstrated that a major reason the downstream site is normally favored is because it occurs at a boundary between A+U rich and non-A+U rich RNA. When the RNA between the two splice sites was made less A+U rich, splicing occurred preferentially at the upstream site.

Gene expression in Leishmania: analysis of essential 5' DNA sequences.

A major unanswered question in Kinetoplastida parasites is the mechanism of regulating gene expression. Using a transfection system, we have previously shown that the intergenic region of the alpha-tubulin gene of Leishmania enriettii contained sequences required for gene expression. The goal of the work reported here was to determine whether the Leishmania-derived sequences were providing transcriptional control signals or functioning at a post-transcriptional level, most likely in trans-splicing. The chloramphenicol acetyltransferase (cat) gene was used as the reporter gene and was stably introduced into L. enriettii as part of an extrachromosomal element by transfection. We show here that the production of cat mRNA was dramatically dependent on the presence of the intergenic region 5' to the cat gene. The intergenic region could be substituted by a smaller fragment (222 base pairs) that contained the trans-splice acceptor site and an adjacent polypyrimidine tract. This native fragment could be replaced by a synthetic polypyrimidine tract containing an AG site. The native and the synthetic fragments had unidirectional activity. No effect on transcription of the cat gene by the wild-type fragment or the synthetic polypyrimidine was detected. The results indicate that both regions contain signals that affect RNA stability, probably sequences involved in trans-splicing.

Cloning and characterization of a gene coding for a protein (KAP) associated with the kinetoplast of epimastigotes and amastigotes of Trypanosoma cruzi

We have cloned and characterized a gene of Trypanosoma cruzi which encodes a protein, KAP (kinetoplasts-associated protein), expressed in the kinetoplasts of epimastigotes and amastigotes, the replicative stages of the parasite, but not in kinetoplasts of trypomastigotes. The single-copy gene is transcribed into a 3900-nt polyadenylated mRNA. Its trans-splicing acceptor site is preceded by a run of 15 adenosine residues. An open reading frame of 1052 codons is followed by a 3′ untranslated region containing short sequences characteristic of rapidly degradable RNAs. The potential translation product of the KAP gene contains a central region composed of four blocks of repeats of a 9-amino-acid motif. Rabbit antibodies raised against three synthetic peptides containing KAP sequence recognized a 175-kDa protein in epimastigotes and amastigotes which appears by indirect immunofluorescence to be associated with their kinetoplasts. The antibodies do not recognize the kinetoplast of trypomastigotes. The amino terminus of KAP contains features compatible with mitochondrial topogenic sequences.