RESUMO
Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Cromossômicas não Histona/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucléolo Celular/metabolismoRESUMO
Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved from archaea to humans and is essential for the transcription initiated by each of the three RNA polymerases. In addition, the discovery of TBP-related factor 1 (TRF1) and other factors related to TBP shed light on the variability among transcription initiation complexes, thus demonstrating that the compositions of these complexes are, in fact, more complicated than originally believed. Despite these facts, the majority of studies on transcription have been performed on animal, plant and fungal cells, which serve as canonical models, and information regarding protist cells is relatively scarce. The aim of this work is to review the diversity of the TBPs that have been documented in protists and describe some of the specific features that differentiate them from their counterparts in higher eukaryotes.
Assuntos
Eucariotos/genética , TATA Box , Proteína de Ligação a TATA-Box , Transcrição Gênica , Eucariotos/metabolismo , Genes de Protozoários , Variação Genética , Giardia/genética , Giardia/metabolismo , Leishmania/genética , Leishmania/metabolismo , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismoRESUMO
The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Protozoários/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Trichomonas vaginalis/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genética , Transcrição Gênica , Trichomonas vaginalis/genéticaRESUMO
Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (TcRPA31-EGFP), we describe its nuclear distribution during growth and metacyclogenesis. Our findings indicate that TcRPA31-EGFP alters its nuclear presence from concentrated nucleolar localization in exponentially growing epimastigotes to a dispersed granular distribution in the nucleoplasm of stationary epimastigotes and metacyclic trypomastigotes. These changes likely reflect a structural redistribution of the Pol I transcription machinery in quiescent cellular stages where downregulation of rRNA synthesis is known to occur. In addition, and related to the nuclear internalization of this protein, the presence of a classical bipartite-type nuclear localization signal was identified towards its C-terminal end. The functionality of this motif was demonstrated by its partial or total deletion in recombinant versions of the tagged fluorescent protein. Moreover, ivermectin inhibited the nuclear localization of the labelled chimaera, suggesting the involvement of the importin α/ß transport system.
Assuntos
Núcleo Celular/enzimologia , Doença de Chagas/metabolismo , Sinais de Localização Nuclear , RNA Polimerase I/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Animais , Regulação para Baixo , RNA Polimerase I/genética , RNA Ribossômico , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanosoma cruzi is a species of parasitic protozoa that causes American trypanosomiasis or Chagas disease. These parasites go through a complex life cycle in Triatominae insects and vertebrate hosts. Epimastigotes are replicative forms that colonize the digestive tract of the vector and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of differences in cells undergoing growth rate transitions from an exponential growth to a stationary phase. Since the classical descriptions of T. cruzi, it has been noted that the growth curve of epimastigotes in culture can give rise, in the stationary phase, to nonreplicating forms of metacyclic trypomastigotes. Metacyclogenesis therefore regards to the development process by which epimastigote transform into infective metacyclic trypomastigotes. In nature, these metacyclic forms allow the spread of Chagas disease when transmitted from an infected vector to a vertebrate host. This work reviews cellular phenomena that occur during the growth rate transitions of epimastigotes in culture, which may be related to very early physiological conditions for metacyclogenesis. Many of these events have not been thoroughly investigated. Their analysis can stimulate new hypotheses and future research in an important area not fully exploited.
Assuntos
Trypanosoma cruzi/citologia , Trypanosoma cruzi/fisiologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180 bp. We also identified a potential upstream control element (UCE) that was located 80 bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34 bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism.
Assuntos
Regiões Promotoras Genéticas , RNA de Protozoário/genética , RNA Ribossômico/genética , Regiões Terminadoras Genéticas , Transcrição Gênica , Trichomonas vaginalis/genética , Sequência de Bases , Genes de Protozoários , Genes de RNAr , Dados de Sequência Molecular , Plasmídeos/genética , RNA Polimerase I/genética , Ribossomos/genética , Sítio de Iniciação de Transcrição , Uridina/genéticaRESUMO
Our group is interested in rRNA and ribosome biogenesis in the parasitic protozoan Trypanosoma cruzi. Epimastigotes represent an extracellular replicative stage of T. cruzi and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of potential differences in the nucleoli of cells undergoing growth-rate transitions. To establish cellular parameters for studying ribosome biogenesis in T. cruzi, a morphometric analysis of the nucleoli of cultured cells in the exponential and stationary phases was conducted. Electron micrograph-based measurements of nuclear sections from independent cells demonstrated that the nucleolar area is over twofold higher in exponentially growing cells, as compared with epimastigotes in the stationary phase. The granular component of the nucleoli of actively growing cells was the main structural element. Cycloheximide moderately reduced the apparent size of the nucleoli without an apparent disruption of their architecture. Our results provide a firm basis for the establishment of an experimental model to study the organization of the nucleolus during the growth and development of T. cruzi.
Assuntos
Trypanosoma cruzi/citologia , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/ultraestrutura , Cicloeximida/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/ultraestruturaRESUMO
The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.
Assuntos
DNA Ribossômico/genética , Eucariotos/genética , Genes de Protozoários , Filogenia , RNA Ribossômico 5S/genética , Animais , Modelos GenéticosRESUMO
The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization.
Assuntos
Genes de Protozoários , RNA Ribossômico 5S/genética , Trichomonas vaginalis/genética , Trichomonas/genética , Tritrichomonas foetus/genética , Animais , Sequência de Bases , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , RNA de Protozoário/química , RNA de Protozoário/genética , RNA Ribossômico 5S/química , Alinhamento de SequênciaRESUMO
A simple model is put forward to explain the long-known three-base periodicity in coding DNA. We propose the concept of same-phase triplet clustering, i.e. a condition wherein a triplet appears several times in one phase without interruption by the two other possible phases. For instance, in the sequence (i): NTT_GNN_NTT_GNN_NTT_GNN_NNN_NTT_GNN (where N is any nucleotide but combinations producing TTG are excluded) there would be clustering of same-phase TTG because this triplet appears uninterruptedly in phase 2. In contrast, in the sequence (ii): TTG_NTT_GNN_NNT_TGN_NNN_NTT_GNN there is no same-phase clustering because neighboring TTGs are all in different phases. Observe also that in sequence (i) TTG triplets are separated by 3, 3 and 6 nucleotides (3n distances), while in sequence (ii) they are separated by 1, 4 and 5 nucleotides (non-3n distances). In this work, we demonstrate that in coding DNA the 3n distances generated by (i)-type sequences proportionally outnumber the non-3n distances generated by (ii)-type sequences, this condition would be the basis of three-base periodicity. Randomized sequences had (i)- and (ii)-type sequences too but clustering was statistically different. To prove our model we generated (i)-type sequences in a randomized sequence by inducing clustering of same-phase triplets. In agreement with the model this sequence displayed three-base periodicity. Furthermore, two- and four-base periodicities could also be induced by artificially inducing clustering of duplets and tetraplets.