A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Abstract

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Fig. 1:. fluxogram illustrating each step of processing and curation performed with sequences from the dbEST and the spliced leader (SL)-enriched Expressed Sequence Tags library. The fluxogram contains both the names of all programs and methods (blue shaded boxes) used in this study for sequence cleaning, validation, trimming and annotation and the results generated by those (pink shaded boxes).

Fig. 2:. pie chart illustrating the distribution of annotated proteins in different classes after manual curation and classification.

Fig. 3:. in vitro cultured Schistosoma mansoni larvae seven days post-double stranded RNA treatments. A, B: brightfield photomicrographs of in vitro cultured S. mansoni sporocysts after seven days of treatments with spliced leader (SL)-small interfering (siRNA) (A) compared to the control decoy-siRNA (B), illustrating the effects of the exposure to SL-siRNA on sporocyst lengths; C: graphic representation of sporocyst length measurements (Î1/4m) after seven days of siRNA treatment by scatter plots with the calculated median values indicated by the horizontal bars. The median values for siRNA treatments were compared to decoy-siRNA (grey plots) treatment control. All mesurements were statistically analysed using Mann-Whitney U test within each experiment. Asterisk means p < 0.0001.

Fig. 4:. histogram depicting the relative transcript levels of small interfering (siRNA)-treated sporocysts after seven days of exposure compared to the decoy-siRNA control. For each transcript tested, data is represented as mean fold-differences (± 2 standard error) relative to the decoy-small RNA control (1.00). Gray bars represent sporocyst messenger RNA levels showing consistent and statistically significant decrease of known trans-spliced transcripts {calcium channel/decoy, p = 0.0056; adenosine triphosphate (ATP)ase inhibitor/decoy, p = 0.0358; phosphoserine-phosphohydrolase/decoy, p = 0.0136; thioredoxin/decoy, p = 0.0358; enolase/decoy, p = 0.0189}. White bars represent relative transcript levels for non-trans-spliced transcripts in siRNA-treated sporocysts that showed no differences when compared to decoy-siRNA treated controls (SmZF1/decoy, p = 0.0755; SOD/decoy, p = 0.8969; SmRBx/decoy, p = 0.0765). Transcript levels were determined by reverse transcriptase-quantitative polymerase chain reaction and data analysed using the Î"Î"Ct method followed by statistical analysis using the Mann-Whitney U test. Significance levels were set at p < 0.05 (*). Data were generated from three independent experiments. The gray line represents the decoy-control level.

Fig. 5:. representative scheme of an alternative spliced leader (SL) trans-splicing, as was observed in the case of ubiquinol-cytochrome C reductase complex ubiquinol binding protein (UbCRBP). While in normal transcription all exons are present in the final transcript, in alternative SL trans-splicing, the SL insertion is between the first and second exons, yielding a shortened transcript with a missing exon.