RESUMO
The pre-mRNA splicing factor PRP19 is recruited into the spliceosome after forming the PRP19/CDC5L complex in humans and the Nineteen complex in yeast. Additionally, 'PRP19-related' proteins enter the spliceosome individually or in pre-assemblies that differ in these systems. The protistan family Trypanosomatidae, which harbors parasites such as Trypanosoma brucei, diverged early during evolution from opisthokonts. While introns are rare in these organisms, spliced leader trans splicing is an obligatory step in mRNA maturation. So far, â¼70 proteins have been identified as homologs of human and yeast splicing factors. Moreover, few proteins of unknown function have recurrently co-purified with splicing proteins. Here we silenced the gene of one of these proteins, termed PRC5, and found it to be essential for cell viability and pre-mRNA splicing. Purification of PRC5 combined with sucrose gradient sedimentation revealed a complex of PRC5 with a second trypanosomatid-specific protein, PRC3, and PRP19-related proteins SYF1, SYF3 and ISY1, which we named PRP19-related complex (PRC). Importantly, PRC and the previously described PRP19 complex are distinct from each other because PRC, unlike PRP19, co-precipitates U4 snRNA, which indicates that PRC enters the spliceosome prior to PRP19 and uncovers a unique pre-organization of these proteins in trypanosomes.
Assuntos
Enzimas Reparadoras do DNA/genética , Proteínas Nucleares/genética , Proteínas de Protozoários/genética , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Interferência de RNA , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Trypanosoma/classificação , Trypanosoma/genética , Trypanosoma/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
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.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , RNA Líder para Processamento/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Poliadenilação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Líder para Processamento/genética , Trans-Splicing/genética , Trypanosoma brucei brucei/genéticaRESUMO
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.
Assuntos
Complexos Multiproteicos/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Spliceossomos , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Imunofluorescência , Espectrometria de Massas , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Splicing de RNA , RNA de Protozoário/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismoRESUMO
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.
Assuntos
Benzoatos/isolamento & purificação , Benzoatos/farmacologia , Piper/química , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Benzoatos/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Estrutura Molecular , Folhas de Planta/química , Raízes de Plantas/química , Caules de Planta/química , Plântula/química , Estereoisomerismo , Tripanossomicidas/químicaRESUMO
Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.
Assuntos
DNA de Protozoário/genética , Precursores de RNA/genética , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Trypanosoma/genética , Sequência de Aminoácidos , Dados de Sequência MolecularRESUMO
The present study reports the Gas Chromatography-Mass Spectrometry (GC-MS) evaluation of the hexanes and dichloromethane fractions from extracts of the red alga Centroceras clavulatum (C. Agardh) Montagne. Twenty three compounds were identified, totaling ca. 42% of both fractions (0.18 g mass extract). The main constituents of the fractions were hexadecanoic acid (17.6%) and pentadecanoic acid (15.9%). Several secondary metabolites with interesting biological activity, such as (-)-loliolide, neophytadiene, phytol were identified. In addition, several classes of secondary metabolites, including phenolic compounds (e.g., phenylacetic acid), terpene derivatives, fatty acids, halogenated compound (e.g., 2-chlorocyclohexenol), lignoids, steroids, esters, amides (e.g., hexadecanamide), ketones, carboxylic acids, aldehydes and alcohols were observed. The occurrence of several of these structural classes is described for the first time in this species. The same fractions analyzed by GC-MS, and a separate set of polar fractions, were evaluated against two life cycle stages (epimastigote and trypomastigote forms) of the protozoan Trypanosoma cruzi and against phytopatogenic fungi Cladosporium cladosporiodes and C. sphaerospermum. The dichloromethane fraction was active against both T. cruzi forms (epimastigote IC(50) = 19.1 µg.mL-1 and trypomastigote IC(50) = 76.2 µg.mL-1). The hexanes and ethyl acetate fractions also displayed activity against both fungi species (200 µg) by TLC-bioautography.
Assuntos
Química Farmacêutica/métodos , Cladosporium/efeitos dos fármacos , Ácidos Graxos/farmacologia , Ácido Palmítico/farmacologia , Extratos Vegetais , Rodófitas/química , Trypanosoma cruzi/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Cladosporium/crescimento & desenvolvimento , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas , Hexanos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cloreto de Metileno/química , Micoses/tratamento farmacológico , Micoses/microbiologia , Ácido Palmítico/química , Extratos Vegetais/análise , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Solventes/química , Especificidade da Espécie , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, 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.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/metabolismo , Capuzes de RNA/metabolismo , RNA Polimerase II/metabolismo , Trypanosoma brucei brucei/enzimologia , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Quinases Ciclina-Dependentes/genética , Técnicas de Silenciamento de Genes , Metilação , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/genética , Interferência de RNA , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA , RNA Líder para Processamento/metabolismo , Transcrição GênicaRESUMO
The phylogenetic relationships among species of Triatoma matogrossensis subcomplex (T. baratai, T. guazu, T. matogrossensis, T. sordida, T. vandae, and T. williami) was addressed by using fragments of cytochrome oxidase I (COI), 16S rDNA (16S), and cytochrome b (Cytb) through Bayesian and parsimony analyses. We did not recover a monophyletic T. matogrossensis subcomplex, and their members were found clustered in three strongly supported clades, as follows: i) T. jurbergi + T. matogrossensis + T. vandae + T. garciabesi + T. sordida; ii) with T. guasayana as the sister group of clade (i); and iii) T. williami + T. guazu, however not closely related to the clade formed by the previously mentioned species. The other two endemic species from Central-Western Brazil, T. baratai and T. costalimai, were not recovered with strong clade support as related to other members of this subcomplex. Results call for a further revision in the classification of the subcomplexes within the genus Triatoma.
Assuntos
Evolução Biológica , DNA Mitocondrial/genética , Triatoma/classificação , Triatoma/genética , Animais , Brasil , Humanos , Especificidade da Espécie , Triatoma/anatomia & histologiaRESUMO
Leishmaniasis and Chagas disease affect millions of people in tropical and subtropical regions. Drugs used currently to treat such diseases often present undesirable side effects and low efficiency. The aim of this work was to identify extracts and isolated compounds from the genus Lippia with leishmanicidal and trypanocidal activity. Fifteen extracts from different plant parts of Lippia species with partially known chemical compositions, four partition fractions, six compounds and a mixture of four interconverting flavanones previously isolated from Lippia salviaefolia and Lippia lupulina were assayed in vitro towards epimastigote forms of Trypanosoma cruzi and promastigote forms of Leishmania amazonensis. The root extract of L. lupulina had potent activity against T. cruzi and L. amazonensis (IC50 of 20.0 and 54.5 µg mL-1, respectively). The triterpenoid oleanonic acid showed the strongest activity against these protozoans (IC50 of 18.5 and 29.9 µM, respectively). Our results indicate that Lippia plants and their derivatives deserve further investigation in the search for new antiprotozoal drugs, particularly for the treatment of leishmaniasis and Chagas disease.
Leishmaniose e doença de Chagas afetam milhões de pessoas em regiões tropicais e subtropicais. As drogas atualmente usadas para tratar estas doenças frequentemente apresentam efeitos colaterais indesejáveis e baixa eficiência. Este trabalho teve como objetivo encontrar extratos, frações e compostos isolados de espécies do gênero Lippia com atividades leishmanicida e tripanocida. Quinze extratos de diferentes partes de plantas do gênero Lippia, com composições químicas parcialmente conhecidas, quatro frações de partição, seis substâncias e uma mistura de quatro flavanonas interconversíveis isolados de Lippia salviaefolia e Lippia lupulina foram testados, in vitro, frente a formas epimastigotas de Trypanosoma cruzi e promastigotas de Leishmania amazonensis. O extrato etanólico das raízes de L. lupulina apresentou atividade potente contra T. cruzi e L. amazonensis (IC50 de 20,0 e 54,5 µg mL-1, respectivamente), enquanto que o ácido oleanônico mostrou as atividades mais fortes contra estes protozoários, com IC50 de 18,5 e 29,9 µM, respectivamente. Estes resultados indicam que espécies do gênero Lippia e seus derivados merecem investigações adicionais na busca por novas terapias antiprotozoárias, especialmente para o tratamento de leishmaniose e doença de Chagas.
Assuntos
Antiprotozoários , Flavonoides/uso terapêutico , Lippia/química , Tripanossomicidas , Ácido Oleanólico/uso terapêutico , Doença de Chagas , Leishmania , Trypanosoma cruziRESUMO
Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.