Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni.

Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo.

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.

One- and two-hybrid analysis of the interactions between components of the Trypanosoma cruzi spliced leader RNA gene promoter binding complex.

The spliced leader (SL) RNA gene promoter is the only RNA polymerase II-dependent promoter characterized to date in trypanosomatids. Transcription of this small nuclear RNA is critical for trypanosomatid cell life because it is needed for polycistronic primary transcripts processing into individual translatable mRNAs. In recent years, a set of divergent fundamental transcription factors required for SL RNA gene transcription have been identified in different trypanosomatids. By means of a yeast two-hybrid system, we analyzed the protein-protein interactions between components of the SL RNA gene promoter binding complex. We also studied the interactions of already described motifs of TATA-binding protein (TBP) and transcription factor II B (TFIIB) orthologs separately. This was followed by investigations of DNA-protein interactions within the SL RNA gene promoter binding complex using one-hybrid analysis. Our results suggest that the complex has two "cores" which contact the promoter DNA, trypanosomal small nuclear RNA activating protein complex (tSNAPc), which has strong interactions between its subunits and a more labile TBP-TFIIA sub-complex.

Sequences involved in mRNA processing in Trypanosoma cruzi.

Gene expression in Trypanosomatids requires processing of polycistronic transcripts to generate monocistronic mRNAs by cleavage events that are coupled to the addition of a Spliced Leader sequence (SL) at the 5'-end and a poly(A) tail at the 3'-end of each mRNA. Here we investigate the sequence requirements involved in Trypanosoma cruzi mRNA processing by mapping all available expressed sequence tags and cDNAs containing poly(A) tail and/or SL to genomic intergenic regions. Amongst other parameters, we determined that the median lengths of 5' untranslated region (UTR) and 3'UTR sequences are 35 and 264 nucleotides, respectively; and that the median distance between SL addition sites and a polypyrimidine motif is 18 nucleotides, whereas the median distance between poly(A) addition sites and the closest polypyrimidine-rich sequence is 40 nucleotides.