RESUMEN
Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.
Asunto(s)
Biosíntesis de Proteínas , ARN Ribosómico , Ribosomas , Saccharomyces cerevisiae , Biosíntesis de Proteínas/genética , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Codón/genéticaRESUMEN
The protozoan parasite Trypanosoma brucei and its disease-causing relatives are among the few organisms that barely regulate the transcription of protein-coding genes. Yet, alterations in its gene expression are essential to survive in different host environments. Recently, tRNA-derived RNAs have been implicated as regulators of many cellular processes within and beyond translation. Previously, we identified the tRNAThr-3'-half (AGU) as a ribosome-associated non-coding RNA able to enhance global translation. Here we report that the tRNAThr-3'-half is generated upon starvation inside the mitochondria. The tRNAThr-3'-half associates with mitochondrial ribosomes and stimulates translation during stress recovery, positively affecting mitochondrial activity and, consequently, cellular energy production capacity. Our results describe an organelle ribosome-associated ncRNA involved in translation regulation to boost the central hub of energy metabolism as an immediate stress recovery response.
Asunto(s)
Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , ARN de Transferencia de Treonina/metabolismo , Ribosomas/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Sensing of environmental cues is crucial for cell survival. To adapt to changes in their surroundings cells need to tightly control the repertoire of genes expressed at any time. Regulation of translation is key, especially in organisms in which transcription is hardly controlled, like Trypanosoma brucei. In this study, we describe the shortening of the bulk of the cellular tRNAs during stress at the expense of the conserved 3' CCA-tail. This tRNA shortening is specific for nutritional stress and renders tRNAs unsuitable substrates for translation. We uncovered the nuclease LCCR4 (Tb927.4.2430), a homologue of the conserved deadenylase Ccr4, as being responsible for tRNA trimming. Once optimal growth conditions are restored tRNAs are rapidly repaired by the trypanosome tRNA nucleotidyltransferase thus rendering the recycled tRNAs amenable for translation. This mechanism represents a fast and efficient way to repress translation during stress, allowing quick reactivation with a low energy input.
Asunto(s)
ARN de Transferencia/metabolismo , Ribonucleasas/metabolismo , Estrés Fisiológico/genética , Trypanosoma brucei brucei/enzimología , Biosíntesis de Proteínas , ARN de Transferencia/química , Trypanosoma brucei brucei/genéticaRESUMEN
In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.
Asunto(s)
Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Ribosomas/genética , Trypanosoma brucei brucei/genética , Polirribosomas/genética , Polirribosomas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia de Treonina/genética , ARN de Transferencia de Treonina/metabolismo , Ribosomas/metabolismo , Estrés Fisiológico , Trypanosoma brucei brucei/metabolismoRESUMEN
The storage of translationally inactive mRNAs in cytosolic granules enables cells to react flexibly to environmental changes. In eukaryotes, Scd6 (suppressor of clathrin deficiency 6)/Rap55 (RNA-associated protein 55), a member of the LSm14 (like-Sm14) family, is an important factor in the formation and activity of P-bodies, where mRNA decay factors accumulate, in stress granules that store mRNAs under adverse conditions and in granules that store developmentally regulated mRNAs. SCD6 from Trypanosoma brucei (TbSCD6) shares the same domain architecture as orthologous proteins in other organisms and is also present in cytosolic granules (equivalent to P-bodies). We show that TbSCD6 is a general repressor of translation and that its depletion by RNAi results in a global increase in protein synthesis. With few exceptions, the steady-state levels of proteins are unchanged. TbSCD6 is not required for the formation of starvation-induced granules in trypanosomes, and unlike Scd6 from yeast, Plasmodium and all multicellular organisms analysed to date, it does not form a complex with the helicase Dhh1 (DExD/H-box helicase 1). In common with Xenopus laevis RAP55, TbSCD6 co-purifies with two arginine methyltransferases; moreover, TbSCD6 itself is methylated on three arginine residues. Finally, a detailed analysis identified roles for the Lsm and N-rich domains in both protein localization and translational repression.
Asunto(s)
Biosíntesis de Proteínas/genética , Proteínas Represoras/fisiología , Ribonucleoproteínas/fisiología , Trypanosoma brucei brucei/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Marcaje Isotópico/métodos , Organismos Modificados Genéticamente , Fenotipo , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Proteoma/análisis , ARN Interferente Pequeño/farmacología , Proteínas Represoras/antagonistas & inhibidores , Ribonucleoproteínas/antagonistas & inhibidores , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T. bruceiâ EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.
Asunto(s)
Secuencias de Aminoácidos , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Factor Tu de Elongación Peptídica/química , Proteínas Protozoarias/química , Ribosomas/metabolismo , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Línea Celular , Complejo IV de Transporte de Electrones/metabolismo , Regulación de la Expresión Génica , Mitocondrias/genética , Proteínas Mitocondriales/fisiología , Datos de Secuencia Molecular , Mutación , Fosforilación Oxidativa , Factor G de Elongación Peptídica/genética , Factor G de Elongación Peptídica/metabolismo , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/fisiología , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Interferencia de ARN , Alineación de Secuencia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.
Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Unión al ARN/metabolismo , Trypanosoma brucei brucei/metabolismo , Dedos de Zinc , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMEN
Trypanosomatids cause widespread disease in humans and animals. Treatment of many of these diseases is hampered by the lack of efficient and safe drugs. New strategies for drug development are therefore urgently needed. It has long been known that the single mitochondrion of trypanosomatids exhibits many unique features. Recently, the mitochondrial translation machinery of trypanosomatids has been the focus of several studies, which revealed interesting variations to the mammalian system. It is the aim of this article to review these unique features and to discuss them in the larger biological context. It is our opinion that some of these features represent promising novel targets for chemotherapeutic intervention that should be studied in more detail.
Asunto(s)
Mitocondrias/metabolismo , Biosíntesis de Proteínas , Trypanosoma/metabolismo , Tripanosomiasis/tratamiento farmacológico , Animales , Aspartato-ARNt Ligasa/metabolismo , Descubrimiento de Drogas , Genoma Mitocondrial , Glucólisis , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Fosforilación Oxidativa , Edición de ARN , ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Trypanosoma/citología , Trypanosoma/efectos de los fármacos , Tripanosomiasis/parasitología , Triptófano-ARNt Ligasa/metabolismoRESUMEN
The parasitic protozoa Trypanosoma brucei has a complex life cycle. Oxidative phosphorylation is highly active in the procyclic form but absent from bloodstream cells. The mitochondrial genome encodes several gene products that are required for oxidative phosphorylation, but it completely lacks tRNA genes. For mitochondrial translation to occur, the import of cytosolic tRNAs is therefore essential for procyclic T. brucei. Whether the same is true for the bloodstream form has not been studied so far. Here we show that the steady-state levels of mitochondrial tRNAs are essentially the same in both life stages. Editing of the imported tRNA(Trp) also occurs in both forms as well as in mitochondria of Trypanosoma evansi, which lacks a genome and a translation system. These results show that mitochondrial tRNA import is a constitutive process that must be mediated by proteins that are expressed in both forms of the life cycle and that are not encoded in the mitochondrial genome. Moreover, bloodstream cells lacking either mitochondria-specific translation elongation factor Tu or mitochondrial tryptophanyl-tRNA synthetase are not viable indicating that mitochondrial translation is also essential in this stage. Both of these proteins show trypanosomatid-specific features and may therefore be excellent novel drug targets.
Asunto(s)
Sangre/parasitología , Mitocondrias/genética , Biosíntesis de Proteínas , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/parasitología , Animales , Animales no Consanguíneos , Transporte Biológico , Femenino , Humanos , Ratones , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Proteínas Protozoarias/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Capping of mRNAs is strictly coupled to RNA polymerase II transcription and there is evidence, mainly from metazoans, that other steps in pre-mRNA processing show a similar linkage. In trypanosomes, however, the mRNA cap is supplied by a trans spliced leader sequence. Thus pre-mRNAs transcribed by RNA Polymerase I are capped by trans splicing, and translation-competent transgenic mRNAs can be produced by RNA Polymerase I and T7 RNA polymerase so long as the primary transcript has a splice acceptor signal. We quantified the efficiency of processing of trypanosome pre-mRNAs produced from a plasmid integrated either at the tubulin locus, or in an rRNA spacer, and transcribed by RNA polymerase II, RNA polymerase I or T7 RNA polymerase. The processing efficiencies were similar for primary transcripts from the tubulin locus, produced by RNA polymerase II, and for RNA from an rRNA spacer, transcribed by RNA polymerase I. Primary transcripts produced by T7 RNA polymerase from the tubulin locus were processed almost as well. There was therefore no evidence for recruitment of the 3'-splicing apparatus by the RNA polymerase. Abundant transcripts transcribed from the rRNA locus by T7 RNA polymerase were somewhat less efficiently processed.
Asunto(s)
Fosfoglicerato Quinasa/genética , Proteínas Protozoarias/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Trans-Empalme , Trypanosoma brucei brucei/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/enzimologíaRESUMEN
The 5'-ends of all Kinetoplastid mRNAs consist of a short sequence added by trans splicing. In contrast to cis splicing in mammals, trans splicing in trypanosomes does not involve sequence-specific recognition of the branch point by the U2 snRNP. In mammalian cells and yeast, U2 snRNP is associated with the multimeric factor SF3b, which contains p14, SF3b10, SF3b14b, SAP49, SAP130, SAP145 and SAP155. The interaction between Trypanosoma cruzi p14 and SAP155 has already been characterised using the yeast 2-hybrid system. We here identify the Trypanosoma brucei RRM-protein DRBD1 as a homologue of SAP49. TAP-tagged DRBD1 co-purified with homologues of p14, SAP130, SAP145 and SAP155. Tagged DRBD1 was found in the nucleus; RNAi targeting DRBD1 inhibited trypanosome growth and caused a very mild splicing defect.
Asunto(s)
Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalmosomas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , Humanos , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Empalmosomas/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNAAsp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNAAsp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNAAsp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNAAsp. This indicates that cytosolic and mitochondrial tRNAAsp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.
Asunto(s)
Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/metabolismo , ARN de Transferencia de Aspártico/biosíntesis , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Adenosina Trifosfato/metabolismo , Animales , Citosol/enzimología , Diseño de Fármacos , Mitocondrias/enzimología , Filogenia , Especificidad por Sustrato , Aminoacilación de ARN de Transferencia/fisiología , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
The eukaryotic exosome is a complex of at least 11 proteins that is required for various 3'-5' exoribonucleolytic RNA processing and degradation reactions. The minimal core consists of 6 RNase PH and 3 S1 domain subunits; various additional proteins may be associated. We describe here the purification of native exosome from Leishmania tarentolae. The yield is sufficient for structural studies of the native exosome. Electron microscopy and image reconstruction of negatively stained preparations revealed the expected six-membered ring structure at 35 A resolution. An additional density suggested that RRP6 and its partner EAP3 (equivalent to Rrp47) might be located at the top of the exosome and at the side of the hexameric ring. No exonuclease or polyadenylation activity was detected in the exosome preparations.
Asunto(s)
Exorribonucleasas/química , Exorribonucleasas/aislamiento & purificación , Leishmania/enzimología , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Animales , Clonación Molecular , Exorribonucleasas/metabolismo , Procesamiento de Imagen Asistido por Computador , Leishmania/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/metabolismoRESUMEN
The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the 'TRAMP' complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.
Asunto(s)
ARN Helicasas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Animales , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
The exosome, a complex of 3'-exoribonucleases and associated proteins, is involved in the degradation of eukaryotic mRNAs in the cytoplasm, and has RNA processing and quality control functions in the nucleus. In yeast, the nuclear exosome differs from the cytoplasmic one in that it contains an additional non-essential component, Rrp6p. In contrast, a small proportion of human RRP6 has been shown to localise to the cytoplasm as well. When we purified the Trypanosoma brucei exosome from cytosolic extracts we found RRP6, apparently in stoichiometric amounts. We here confirm that RRP6 is in the trypanosome cytoplasm and nucleus. The level of RRP6 was unaffected by depletion of core exosome subunits by RNA interference and over-expression of tagged RRP6 was possible, indicating that RRP6 can be present independent of exosome association.
Asunto(s)
Exorribonucleasas/metabolismo , Membranas Intracelulares/enzimología , Trypanosoma brucei brucei/enzimología , Animales , Línea Celular , Núcleo Celular/enzimología , Citosol/enzimología , Exorribonucleasas/genética , Mutación/genética , Unión Proteica , Interferencia de ARN , Trypanosoma brucei brucei/genéticaRESUMEN
Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Relación Dosis-Respuesta a Droga , Ácido Edético/química , Electroforesis en Gel de Poliacrilamida , Genotipo , Glutatión Transferasa/metabolismo , Hidrólisis , Inmunoprecipitación , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Microscopía de Contraste de Fase , Modelos Biológicos , Ácido Palmítico/metabolismo , Unión Proteica , Proteínas R-SNARE , Proteínas Recombinantes/química , Proteínas SNARE , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Tiempo , Vacuolas/metabolismoRESUMEN
The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian 'bloodstream form' trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.