Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Abstract

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independentIn the process of pre-mRNA maturation, known as splicing, the introns (non-coding regions of polypeptides) are removed from primary transcripts and the exons (coding regions) joined to form a contiguous sequence specifying a functional polypeptide. It was observed that the process is dependent on ribonucleoproteins, small molecules of RNA binding proteins with different forms and sizes, assembling a macromolecular complex called the spliceosome, where the maturation reaction takes place (Leningher et al. 1995). In trypanosomes, all the RNAs are processed by a mechanism different from that in mammals (cis-splicing) called trans-splicing, a process that involves the union of the exons in two independent transcripts. However, in recent studies it was observed that cis-splicing can also occur in the gene PAP (poly-A polymerase) of Trypanosoma brucei and T. cruzi, suggesting that both trans and cis-splicing, occur in these parasites, with a predominance of trans-splicing (Mair et al. 2000).The SL RNA or miniexon is a primary transcript synthesized independently of pre-mRNA and trans-spliced in all nuclear mRNA. The majority of mRNAs is synthesized as polycistronic precursors (Agabian 1990). T. brucei SL RNA contains an unusual cap structure consisting of 7-methylguanosine linked to four modified nucleosides. This modification of trypanosomatid SL sequence might be required for the function of the SL RNA in trans-splic-

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

“…These results are compatible with those reported by Ambrosio et al (19) and in addition suggest that somehow trans-splicing can also be affected in these parasites, since some RNA bands in the gels are related to the small nuclear RNAs involved in this mechanism (20).…”

“…However, the results obtained thus far show that this interference still promotes the trans-splicing reaction in the cells but at a lower rate than observed in the control, indicating that the drug activity did not completely abolish the trans-splicing reaction (19).…”

“…The permeable cell reactions were carried out according to Ullu and Tschudi (18) and Ambrosio et al (19). In the experiments, 1.25 x 10 8 T. cruzi epimastigote forms (Y, NCS and BOL strains) were washed and resuspended in TB (1X) solution (20 mM KGlu, 3 mM MgCl 2 , 1 mM DTT, 10 µg/mL leupeptin) and treated with 100 µg L-α-lysophosphatidylcholine and palmitoyl (Lysolecithin, Sigma, St. Louis, MO, USA).…”

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

“…Trans-splicing participates in the processing of polycistronic transcription units into individual mRNAs in which a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor premRNA, giving origin to the mature mRNA. We showed that NFOH-121 affected the RNA processing and the trans-splicing reaction, even though at lower intensity (19).…”

Tools for Trans-Splicing Drug Interference Evaluation in Kinetoplastid

“…Ambrósio and co-workers (2004) demonstrated that standardization of trans-splicing reaction with T. cruzi epimastigote forms (Y strain) permeable cells facilitates the study of RNA processing in these trypanosomatids and can be used as a model of study drug activity on trans splicing mechanism. The authors also demonstrated that pro-drug NFOH-121, derived from nitrofurans (Chung et al, 2003), was able to interfere with RNA processing this parasite, although the reaction of trans-splicing has not been affected (Ambrósio et al, 2004). The nitrofurans are compounds with the capacity to inhibit trypanotiona reductase, an important enzyme involved in the antioxidant metabolism of T. cruzi.…”