RESUMEN
Regulation of gene expression in trypanosomatid parasitic protozoa is mainly achieved posttranscriptionally. RNA-binding proteins (RBPs) associate to 3' untranslated regions in mRNAs through dedicated domains such as the RNA recognition motif (RRM). Trypanosoma cruzi UBP1 (TcUBP1) is an RRM-type RBP involved in stabilization/degradation of mRNAs. TcUBP1 uses its RRM to associate with cytoplasmic mRNA and to mRNA granules under starvation stress. Here, we show that under starvation stress, TcUBP1 is tightly associated with condensed cytoplasmic mRNA granules. Conversely, under high nutrient/low density-growing conditions, TcUBP1 ribonucleoprotein (RNP) complexes are lax and permeable to mRNA degradation and disassembly. After dissociating from mRNA, TcUBP1 can be phosphorylated only in unstressed parasites. We have identified TcP22, the ortholog of mammalian P32/C1QBP, as an interactor of TcUBP1 RRM. Overexpression of TcP22 decreased the number of TcUBP1 granules in starved parasites in vivo. Endogenous TcUBP1 RNP complexes could be dissociated in vitro by addition of recombinant TcP22, a condition stimulating TcUBP1 phosphorylation. Biochemical and in silico analysis revealed that TcP22 interacts with the RNA-binding surface of TcUBP1 RRM. We propose a model for the decondensation of TcUBP1 RNP complexes in T. cruzi through direct interaction with TcP22 and phosphorylation.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Extractos Celulares , Humanos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Simulación del Acoplamiento Molecular , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas Recombinantes , Inanición/metabolismo , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Trypanosomes are unique eukaryotic cells, in that they virtually lack mechanisms to control gene expression at the transcriptional level. These microorganisms mostly control protein synthesis by posttranscriptional regulation processes, like mRNA stabilization and degradation. Transcription in these cells is polycistronic. Tens to hundreds of protein-coding genes of unrelated function are arrayed in long clusters on the same DNA strand. Polycistrons are cotranscriptionally processed by trans-splicing at the 5' end and polyadenylation at the 3' end, generating monocistronic units ready for degradation or translation. In this work, we show that some trans-splicing/polyadenylation sites may be skipped during normal polycistronic processing. As a consequence, dicistronic units or monocistronic transcripts having long 3' UTRs are produced. Interestingly, these unspliced transcripts can be processed into mature mRNAs by the conventional trans-splicing/polyadenylation events leading to translation. To our knowledge, this is a previously undescribed mRNA maturation by trans-splicing uncoupled from transcription. We identified an RNA-recognition motif-type protein, homologous to the mammalian polypyrimidine tract-binding protein, interacting with one of the partially processed RNAs analyzed here that might be involved in exon skipping. We propose that splice-site skipping might be part of a posttranscriptional mechanism to regulate gene expression in trypanosomes, through the generation of premature nontranslatable RNA molecules.
Asunto(s)
Poliadenilación , Procesamiento Postranscripcional del ARN , ARN Protozoario/genética , Trans-Empalme , Trypanosoma/genética , Regiones no Traducidas 3' , Animales , Regulación de la Expresión Génica , Genes ProtozoariosRESUMEN
Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.
Asunto(s)
Carioferinas/metabolismo , Transporte de ARN , ARN Protozoario/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Trypanosoma cruzi/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Antiprotozoarios/metabolismo , Antiprotozoarios/farmacología , División Celular , Núcleo Celular/metabolismo , Clonación Molecular , Secuencia Conservada/genética , ADN Protozoario/química , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Carioferinas/genética , Datos de Secuencia Molecular , Poli A/metabolismo , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Análisis de Secuencia de ADN , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiología , Proteína Exportina 1RESUMEN
Trypanosomes, protozoan parasites of the order Kinetoplastida, control gene expression essentially through post-transcriptional mechanisms. Several motifs located mainly in the 3' untranslated region, such as AU-rich elements (AREs), were recently shown to modulate mRNA half-life, and are able to modify mRNA abundance in vivo through the interaction with specific RNA-binding proteins. Along with the detection of an active exosome, decapping activities and a regulated 3' to 5' exonuclease activity stimulated by AREs, these results suggest that modulation of mRNA stability is essential in trypanosomes. These regulatory processes are specific for different developmental stages and thus relevant for allowing trypanosomes to adapt to variable environmental conditions.
Asunto(s)
Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Proteínas de Unión al ARN/fisiología , Trypanosoma/genética , Animales , Regulación de la Expresión Génica , Modelos Genéticos , ARN Mensajero/genética , Proteínas de Unión al ARN/clasificación , Transcripción Genética/genética , Trypanosoma/clasificación , Trypanosoma/metabolismoRESUMEN
Trypanosomes, protozoan parasites from the order Kinetoplastida, have to deal with environmental changes during the interaction with their hosts. Trypanosoma cruzi, the causative agent of Chagas' disease, uses post-transcriptional mechanisms to regulate gene expression. However, few RNA-binding proteins involved in mRNA turnover control have been identified to date. In this work, an RNA recognition motif (RRM)-type RNA-binding protein family named T. cruzi RNA-binding protein (TcRBP) and composed of at least six members was identified. The genomic organization of four members revealed a head to tail arrangement within a region of 15 kilobase pairs. TcRBP members have a common RRM and different auxiliary domains with a high content of glycine, glutamine, and histidine residues within their N- and C-terminal regions. TcRBPs differ in their expression patterns as well as in their homoribopolymer binding interaction in vitro, although they preferentially recognize poly(U) and poly(G) RNAs. An interesting observation was the relaxed RNA-binding interactions with several trypanosome transcripts in vitro. In contrast, co-immunoprecipitation experiments of TcRBP-containing ribonucleoprotein complexes formed in vivo revealed a highly restricted binding interaction with specific RNAs. Several TcRBP-containing complexes are stage-specific and, in some cases, bear the poly(A)-binding protein TcPABP1. Altogether, these results suggest that TcRBPs might be modulated in vivo, to favor or preclude the interaction with specific transcripts in a developmentally regulated manner.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , ARN/metabolismo , Trypanosoma cruzi/química , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , ADN/química , Enzimas de Restricción del ADN/metabolismo , Glutamina/análisis , Glicina/análisis , Histidina/análisis , Datos de Secuencia Molecular , Filogenia , Poli G/metabolismo , Poli U/metabolismo , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/análisis , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Trypanosomes, protozoan parasites causing worldwide infections in human and animals, mostly regulate protein expression through post-transcriptional mechanisms and not at the transcription initiation level. We have previously identified a Trypanosoma cruzi RNA-binding protein named TcUBP-1. This protein is involved in mRNA destabilization in vivo through binding to AU-rich elements in the 3'-untranslated region of SMUG mucin mRNAs (D'Orso, I., and Frasch, A. C. (2001) J. Biol. Chem. 276, 34801-34809). In this work we show that TcUBP-1 is part of an approximately 450-kDa ribonucleoprotein complex with a poly(A)-binding protein and a novel 18-kDa RNA-binding protein, named TcUBP-2. Recombinant TcUBP-1 and TcUBP-2 proteins recognize U-rich RNAs with similar specificity and affinity through the approximately 92-amino acid RNA recognition motif. TcUBPs can homo- and heterodimerize in vitro through the glycine-rich C-terminal region. This interaction was also detected in vivo by co-immunoprecipitation of the ribonucleoprotein complex and using yeast two-hybrid assay. The poly(A)-binding protein identified was shown to disrupt the formation of TcUBP-1, but not TcUBP-2, homodimers in vitro. The possible role of TcUBP-1 ligands in the pathways that govern mRNA-stability and stage-specific expression in trypanosomes is discussed.