In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans-and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.Trypanosomatid parasites utilize RNA splicing for the maturation of nuclear pre-mRNA in two distinct ways: first, as in other eukaryotes, cis splicing is used for intron removal. While this was unambiguously demonstrated for the Trypanosoma brucei poly(A) polymerase (PAP) mRNA (19), intron removal appears to be a rare event in trypanosomatids because a survey of the Tritryp genomes has identified only three additional putative intron-containing genes (10). Second, trypanosomatids process all of their nuclear pre-mRNAs by spliced leader (SL) trans splicing (reviewed in reference 15). In this splicing reaction, the capped, 39 nucleotide (nt)-long 5Ј terminus of the SL RNA, the SL or miniexon, is fused to the 5Ј end of each mRNA. This process is therefore a posttranscriptional mRNA capping mechanism and, since trypanosomatids transcribe their protein coding genes polycistronically, it resolves individual mRNAs from polycistronic precursors in conjunction with polyadenylation. SL trans splicing occurs in several organisms, including tunicate chordates, nematodes, and trematodes, but it is not found in insect and mammalian cells; thus, it is specific to the parasites and not present in the hosts of trypanosomatids. Hence, factors with specific fun...

, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m 7 G capping enzymes. The first critical step, Ser 5 phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m 7 G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

The flagellate protozoan Trypanosoma cruzi, the etiological agent of Chagas disease, affects more than 18 million people in Latin America and is responsible for approximately 400000 deaths per year.1) Only two drugs are commercially available for the treatment of this disease, namely, nifurtimox (a 2-nitrofuran derivative) and benznidazole (a 2-nitroimidazole acetamide). These drugs are, however, not consistently effective and, moreover, exhibit serious side effects including cardiac and/or renal toxicity.2) There is thus particular interest in the discovery of natural products that might be developed to generate safer and more efficient chemotherapeutic agents against T. cruzi. 3)A number of biologically-active chromenes have been isolated from species of the genus Piper, including the prenylated chromene 1 from Piper gaudichaudianum 4) and chromenes 2-5 ( Fig. 1) from P. aduncum. 5) Whilst these chromenes have been shown to exhibit anti-fungal 4) and antitumour properties, 5) no evaluation has yet been made with respect to their activity against T. cruzi. The object of the present study was to examine a range of natural chromenes and chromene derivatives in order to determine their potential for further development to treat Chagas disease. METHODS AND RESULTSGeneral All reagents were of analytical grade. For methylation reactions, an ethereal solution of diazomethane (30 ml) was prepared by dissolving 2.14 g of N-methyl-N-nitroso-p-toluenesulphonamide (Diazald; Aldrich, Steinheim, Germany) in 10.0 ml of ethanol containing 4.0% potassium hydroxide.6) Hydrogenation reactions were carried out under an atmosphere of hydrogen in the presence of palladium (3.0 or 10.0%) on activated charcoal (Acros Organics, New Jersey, U.S.A.) as catalyst.7) The acetylated chromene was prepared by treatment with acetic anhydride (20.0 ml) and pyridine (20.0 ml) overnight. 8) Benznidazole, employed as positive control in the assays of trypanocidal activity, was obtained from Roche (Rio de Janeiro, Brazil).Plant Material Specimens of P. gaudichaudianum and P. aduncum were cultivated from seed under greenhouse conditions at the Institute of Chemistry, UNESP, Araraquara-SP, Brazil. Plant material was authenticated by Dr. Guillermo E. D. Paredes (Universidad Pedro Ruiz Gallo, Lambayeque, Peru) and voucher specimens (with codes Kato 0093 and 0057, respectively) were deposited at the Herbarium of the The aim of the study was to investigate the anti-trypanocidal activities of natural chromene and chromene derivatives. Five chromenes were isolated from Piper gaudichaudianum and P. aduncum, and a further seven derivatives were prepared using standard reduction, methylation and acetylation procedures. These compounds were assayed in vitro against epimastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. The results showed that the most of the compounds, especially those possessing electron-donating groups as substituents on the aromatic ring, showed potent trypanocidal activity. The most active compound, [(2S)-methyl-2-m...

Gaudichaudianic acid, a prenylated chromene isolated from Piper gaudichaudianum, has been described as a potent trypanocidal compound against the Y-strain of Trypanosoma cruzi. We herein describe its isolation as a racemic mixture followed by enantiomeric resolution using chiral HPLC and determination of the absolute configuration of the enantiomers as (+)-S and (-)-R by means of a combination of electronic and vibrational circular dichroism using density functional theory calculations. Investigation of the EtOAc extract of the roots, stems, and leaves from both adult specimens and seedlings of P. gaudichaudianum revealed that gaudichaudianic acid is biosynthesized as a racemic mixture from the seedling stage onward. Moreover, gaudichaudianic acid was found exclusively in the roots of seedlings, while it is present in all organs of the adult plant. Trypanocidal assays indicated that the (+)-enantiomer was more active than its antipode. Interestingly, mixtures of enantiomers showed a synergistic effect, with the racemic mixture being the most active.

Summary In trypanosomes, mRNAs are processed by spliced leader (SL) trans splicing, in which a capped SL, derived from SL RNA, is spliced onto the 5′ end of each mRNA. This process is mediated by the spliceosome, a large and dynamic RNA-protein machinery consisting of small nuclear ribonucleoproteins (snRNPs) and non-snRNP proteins. Due to early evolutionary divergence, the amino acid sequences of trypanosome splicing factors exhibit limited similarity to those of their eukaryotic orthologs making their bioinformatic identification challenging. Most of the ~ 60 protein components that have been characterized thus far are snRNP proteins because, in contrast to individual snRNPs, purification of intact spliceosomes has not been achieved yet. Here, we characterize the non-snRNP PRP19 complex of Trypanosoma brucei. We identified a complex that contained the core subunits PRP19, CDC5, PRL1, and SPF27, as well as PRP17, SKIP and PPIL1. Three of these proteins were newly annotated. The PRP19 complex was associated primarily with the activated spliceosome and, accordingly, SPF27 silencing blocked the first splicing step. Interestingly, SPF27 silencing caused an accumulation of SL RNA with a hypomethylated cap that closely resembled the defect observed previously upon depletion of the cyclin-dependent kinase CRK9, indicating that both proteins may function in spliceosome activation.

In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 ablation was lethal in cultured trypanosomes, causing a block of trans splicing before the first transesterification step. Additionally, CRK9 silencing led to dephosphorylation of RNA polymerase II and to hypomethylation of the SL cap structure. Here, we tandem affinity-purified CRK9 and, among potential CRK9 substrates and modifying enzymes, discovered an unusual tripartite complex comprising CRK9, a new L-type cyclin (CYC12) and a protein, termed CRK9-associated protein (CRK9AP), that is only conserved among kinetoplastids. Silencing of either CYC12 or CRK9AP reproduced the effects of depleting CRK9, identifying these proteins as functional partners of CRK9 in vivo. While mammalian cyclin L binds to CDK11, the CRK9 complex deviates substantially from that of CDK11, requiring CRK9AP for efficient CRK9 complex formation and autophosphorylation in vitro. Interference with this unusual CDK rescued mice from lethal trypanosome infections, validating CRK9 as a potential chemotherapeutic target.

The trypanocidal activity of crude extracts and fractions from the leaves and stems of Peperomia obtusifolia (Piperaceae) was evaluated in vitro against the epimastigote forms of Trypanosoma cruzi. Bioactivity-guided fractionation of the most active extracts afforded seven known compounds, including three chromanes, two furofuran lignans and two flavone C-diglycosides. The most active compounds were the chromanes peperobtusin A and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(2''-methyl-2''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2 H-1-benzopyran-6-carboxylic acid, with IC (50) values of 3.1 microM (almost three times more active than the positive control benznidazole, IC (50) 10.4 microM) and 27.0 microM, respectively. Cytotoxicity assays using peritoneal murine macrophages indicated that the chromanes were not toxic at the level of the IC (50) for trypanocidal activity. This is the first report on the trypanocidal activity besides unspecific cytotoxicity of chromanes from Peperomia species. Additionally it represents the first time isolation of 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(2''-methyl-2''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2 H-1-benzopyran-6-carboxylic acid from P. obtusifolia.

SUMMARYA study was conducted of the biological, morphological and molecular characters of 3 strains of Trypanosoma cruzi (SI5, SI8 and SIGR3) isolated from specimens of Triatoma sordida collected in Santo Inácio and a domestic cat. In order to carry out the study, the following parameters were evaluated: pre-patent period, parasitaemia curves, morphology of the parasites, mortality rates, histopathological lesions and molecular typing. The strains presented variable pre-patent periods, low parasitaemia and no animal mortality. The morphological study of trypomastigotes showed a predominance of intermediate-width and short-length forms, as well as low nuclear index. Epimastigotes presented a low nuclear index, intermediate-width forms in strains SI5 and SI8, and large-width forms in SIGR3. A shorter length could be noted in strains SI8 and SIGR3, whereas SI5 displayed an intermediate length. The histopathological study did not detect amastigote nests in tissues. The amplification of the divergent domain of 24Sα rRNA, HSP60 and GPI genes of strains SI5, SI8 and SIGR3 classified the 3 strains into Group II. Biological parameters made it possible to classify the strains isolated in Santo Inácio (BA) into Biodeme III, Zymodeme 1 and Group II of T. cruzi.