RESUMEN
Unicellular flagellates of the family Trypanosomatidae are obligatory parasites of invertebrates, vertebrates and plants. Dixenous species are aetiological agents of a number of diseases in humans, domestic animals and plants. Their monoxenous relatives are restricted to insects. Because of the high biological diversity, adaptability to dramatically different environmental conditions, and omnipresence, these protists have major impact on all biotic communities that still needs to be fully elucidated. In addition, as these organisms represent a highly divergent evolutionary lineage, they are strikingly different from the common 'model system' eukaryotes, such as some mammals, plants or fungi. A number of excellent reviews, published over the past decade, were dedicated to specialized topics from the areas of trypanosomatid molecular and cell biology, biochemistry, host-parasite relationships or other aspects of these fascinating organisms. However, there is a need for a more comprehensive review that summarizing recent advances in the studies of trypanosomatids in the last 30 years, a task, which we tried to accomplish with the current paper.
Asunto(s)
Evolución Biológica , Regulación de la Expresión Génica , Genoma de Protozoos , Filogenia , Trypanosomatina , Animales , Regulación de la Expresión Génica/genética , Humanos , Trypanosomatina/clasificación , Trypanosomatina/genética , Trypanosomatina/metabolismoRESUMEN
Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3' adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation.
Asunto(s)
Factores de Transcripción Activadores/genética , Mitocondrias/genética , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Factores de Transcripción Activadores/metabolismo , Animales , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Poliadenilación , Biosíntesis de Proteínas , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas/genética , Subunidades Ribosómicas Pequeñas/metabolismo , Ribosomas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
The mitochondrial 45 S SSU* complex in Trypanosoma brucei contains the 9 S SSU ribosomal RNA, a set of SSU ribosomal proteins, several pentatricopeptide repeat (PPR) proteins, and proteins not typically found in ribosomes, including rhodanese domain protein (Rhod) and a 200-kDa coiled-coil protein. To investigate the function of this complex, PPR29, Rhod, 200-kDa protein, and mitochondrial ribosomal protein S17 were knocked down by RNAi in procyclic T. brucei. A growth retardation phenotype, a reduction in the amount of the 45 S SSU* complexes, and the preferential inhibition of synthesis of the cytochrome c oxidase subunit I over apocytochrome b were observed as early as day 2 postinduction of RNAi. On the contrary, the down-regulation of mitochondrial ribosomal protein L3 drastically reduced the amount of the large subunit and indiscriminately inhibited mitochondrial translation. The relative amounts of translation-competent, long poly(AU)-tailed cytochrome c oxidase subunit I and edited apocytochrome b mRNAs were selectively reduced by ablation of the 45 S SSU* complex. The formation of the 80 S translation complexes, identified by association of the long-tailed mRNAs with the mitoribosomes, was also disrupted. On the other hand, the relative amount of long-tailed edited RPS12 mRNA was not substantially affected, and there was no noticeable effect on the RPS12 translation complexes. In bloodstream trypanosomes, the amount of the 45 S complexes was drastically reduced compared with procyclics. We propose that the 45 S SSU* complex represents a factor required for normal mitochondrial translation that may have selective effects on different mRNAs.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Protozoarias/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo , Trypanosoma brucei brucei/metabolismo , Técnicas de Silenciamiento del Gen , Mitocondrias/genética , Proteínas Mitocondriales/genética , Complejos Multiproteicos/genética , Proteínas Protozoarias/genética , Edición de ARN/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs. The genetic information for editing resides in small guide RNAs (gRNAs), which form anchor duplexes just downstream of an editing site and mediate editing within a single editing "block." Many mRNAs require multiple gRNAs; the observed overall 3' to 5' polarity of editing is determined by the formation of upstream mRNA anchors by downstream editing. Hel61, a mitochondrial DEAD-box protein, was previously shown to be involved in RNA editing, but the functional role was not clear. Here we report that down-regulation of Hel61 [renamed REH1 (RNA editing helicase 1)] expression in Trypanosoma brucei selectively affects editing mediated by two or more overlapping gRNAs but has no effect on editing within a single block. Down-regulation produces an increased abundance of the gRNA/edited mRNA duplex for the first editing block of the A6 mRNA. Recombinant REH1 has an ATP-dependent double strand RNA unwinding activity in vitro with a model gRNA-mRNA duplex. These data indicate that REH1 is involved in gRNA displacement either directly by unwinding the gRNA/edited mRNA duplex or indirectly, to allow the 5' adjacent upstream gRNA to form an anchor duplex with the edited mRNA to initiate another block of editing. Purified tagged REH1 is associated with the RNA editing core complex by RNA linkers and a colocalization of REH1, REL1, and two kinetoplast ribosomal proteins with the kinetoplast DNA was observed by immunofluorescence, suggesting that editing, transcription, and translation may be functionally linked.
Asunto(s)
Mutación INDEL/genética , Proteínas Protozoarias/metabolismo , Edición de ARN/genética , ARN Helicasas/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Trypanosoma brucei brucei/enzimología , Uridina/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Regulación hacia Abajo/genética , Espacio Intracelular/metabolismo , Mitocondrias/metabolismo , Transporte de Proteínas , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.
Asunto(s)
ADN de Cinetoplasto/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Edición de ARN , ARN Ribosómico/metabolismo , Trypanosoma brucei brucei/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genoma de Protozoos , Proteínas Mitocondriales/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genéticaRESUMEN
Several new species of trypanosomatids (Euglenozoa, Kinetoplastea, Trypanosomatidae), isolated from the intestines of Neotropical insects (Heteroptera), were genotyped on the basis of spliced leader RNA, and also defined phylogenetically using gene sequences of small subunit ribosomal RNA and glycosomal glyceraldehyde phosphate dehydrogenase. The taxonomic descriptions also included characterization using morphometry and electron microscopy. Our phylogenetic analyses placed the new species within the clade, previously designated "SE" for "Slowly Evolving" sequences of ribosomal RNA genes, a clade that also includes numerous monoxenous parasites of insects from the genera Crithidia, Leptomonas, and Wallaceina, as well as the dixenous genus Leishmania. Based on the high phylogenetic support for this clade, which is consistently recovered in all recent phylogenetic reconstructions, a proposal is put forward to recognize this natural taxon as a new subfamily, Leishmaniinae, within the family Trypanosomatidae.
Asunto(s)
Insectos/parasitología , Trypanosomatina/clasificación , Trypanosomatina/aislamiento & purificación , Animales , Análisis por Conglomerados , Costa Rica , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Microscopía , Datos de Secuencia Molecular , Filogenia , ARN Protozoario/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN , Trypanosomatina/genéticaRESUMEN
Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.
Asunto(s)
Basidiomycota/clasificación , Basidiomycota/aislamiento & purificación , Heterópteros/microbiología , Animales , Basidiomycota/genética , Basidiomycota/fisiología , Análisis por Conglomerados , Costa Rica , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Intestinos/microbiología , Datos de Secuencia Molecular , Filogenia , Saccharomycetales , Análisis de Secuencia de ADNRESUMEN
The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes).
Asunto(s)
Leishmania/genética , Mitocondrias/química , Ribosomas/química , Animales , Microscopía por Crioelectrón , Mitocondrias/metabolismo , Modelos Moleculares , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Flagellates of the class Kinetoplastea are known to frequently parasitize insects. We have collected 67 isolates from 407 Heteroptera hosts captured in several locations of South-West China. Their splice leader (SL) RNA gene repeats and small subunit (SSU) rRNA genes were PCR amplified from the infected tissue samples. In most cases, parasites were found in the midgut, rarely the infection was confined to the Malpighian tubes. Phylogenetic analysis of the obtained sequences has significantly expanded the known diversity of these monoxenous parasites. Fifteen typing units were found among these isolates including 11 potentially new species. Four typing units matched the previously known typing units from the Neotropics indicating a global distribution of the respective parasite species. At the same time, new clades appeared, testifying for a certain level of endemism. The host record of the parasites found indicated a variable specificity level of the host-parasite association including several cases of a very broad host range. Our results disprove the "one host - one parasite" paradigm and show that although the global diversity of monoxenous parasites is high, it is not as enormous as suggested earlier. Moreover, phylogenetic analysis revealed the presence, among the isolated strains, of a new Phytomonas species, which is the first documentation of this potentially pathogenic dixenous parasite of plants in China.
Asunto(s)
Evolución Molecular , Insectos/parasitología , Filogenia , Trypanosomatina/genética , Animales , China , ADN Protozoario/genética , Genes Protozoarios , Genes de ARNr , Geografía , Interacciones Huésped-Parásitos , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la Especie , Trypanosomatina/clasificaciónRESUMEN
Two new trypanosomatid species (Euglenozoa, Kinetoplastea) isolated from the intestinal tract of heteropteran insect hosts were described based on molecular phylogenetic analyses of Spliced Leader (SL) RNA gene repeats, glycosomal glyceraldehyde phosphate dehydrogenase, and small subunit ribosomal RNA genes, as well as by morphology. Leptomonas barvae n. sp., from a mirid host Collaria oleosa, was found to represent one of the closest monoxenous (one host) relatives of the dixenous (two hosts) parasitic genus Leishmania. This finding further supports the origin of these dixenous parasites from monoxenous progenitors in the Neotropics. Blastocrithidia largi n. sp., from a largid host Largus cinctus, is among a few members of this genus available in culture. The species is a close relative of Blastocrithidia triatomae and is a member of a new monophyletic phylogenetic group characterized by formation of straphanger cysts.
Asunto(s)
Heterópteros/parasitología , Trypanosoma/clasificación , Trypanosoma/aislamiento & purificación , Animales , Análisis por Conglomerados , Costa Rica , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Tracto Gastrointestinal/parasitología , Genes de ARNr , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Microscopía , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/genética , ARN Protozoario/genética , ARN Ribosómico 18S/genética , ARN Lider Empalmado/genética , Análisis de Secuencia de ADN , Trypanosoma/citología , Trypanosoma/genéticaRESUMEN
Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.
Asunto(s)
Edición de ARN/fisiología , ARN Mitocondrial/metabolismo , ARN Protozoario/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , ARN Mitocondrial/genética , ARN Protozoario/genética , Trypanosoma brucei brucei/genéticaRESUMEN
In the mitochondria of trypanosomes and related kinetoplastid protists, most mRNAs undergo a long and sophisticated maturation pathway before they can be productively translated by mitochondrial ribosomes. Some of the aspects of this pathway (identity of the promotors, transcription initiation, and termination signals) remain obscure, and some (post-transcriptional modification by U-insertion/deletion, RNA editing, 3'-end maturation) have been illuminated by research during the last decades. The RNA editing creates an open reading frame for a productive translation, but the fully edited mRNA often represents a minor fraction in the pool of pre-edited and partially edited precursors. Therefore, it has been expected that the final stages of the mRNA processing generate molecular hallmarks, which allow for the efficient and selective recognition of translation-competent templates. The general contours and several important details of this process have become known only recently and represent the subject of this review.
RESUMEN
In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae, with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and differences to human-pathogenic species, as these aspects are relevant for the evaluation of biosafety aspects of L. tarentolae as host for recombinant DNA/protein applications. Studies indicate that strain LEM-125 but not strain TARII/UC of L. tarentolae might also be capable of infecting mammals, at least transiently. This could raise the question of whether the current biosafety level of this strain should be reevaluated. In addition, we will summarize the current state of biotechnological research involving L. tarentolae and explain why this eukaryotic parasite is an advantageous and promising human recombinant protein expression host. This summary includes overall biotechnological applications, insights into its protein expression machinery (especially on glycoprotein and antibody fragment expression), available expression vectors, cell culture conditions, and its potential as an immunotherapy agent for human leishmaniasis treatment. Furthermore, we will highlight useful online tools and, finally, discuss possible future applications such as the humanization of the glycosylation profile of L. tarentolae or the expression of mammalian recombinant proteins in amastigote-like cells of this species or in amastigotes of avirulent human-pathogenic Leishmania species.
Asunto(s)
Biotecnología/métodos , Leishmania/clasificación , Proteínas Recombinantes/biosíntesis , Animales , Glicosilación , Humanos , Leishmania/patogenicidad , Leishmaniasis , Procesamiento Proteico-PostraduccionalRESUMEN
Three new species of monoxenous parasites from the Neotropical Heteroptera are described on the basis of the ultrastructure of cells in culture, as well as gene sequences of Spliced Leader (SL) RNA, glyceraldehyde phosphate dehydrogenase (GAPDH) and small subunit (SSU) rRNA. The results have highlighted a striking discrepancy between the morphological (dis)similarities and the phylogenetic affinities among the insect trypanosomatids. Although each of the new species is characterized by a distinct set of morphological characters, based on the predominant promastigotes observed in culture, each of them has been provisionally assigned to the genus Leptomonas pending the future revision of this genus. Yet, instead of the phylogenetic affinity with the other members of this polyphyletic genus, the new species are most closely related to Crithidia species. Thus, the extremely long promastigotes of Leptomonas acus sp. n. and the unique morphological features found in Leptomonas bifurcata sp. n. sharply contrast with their respective relatives C. fasciculata and C. deanei both of which are typical choanomastigotes. The results clearly show that the current classification at the genus level is misleading and needs to be revised. The phylogenetic clades potentially representing the candidate new genera of monoxenous trypanosomatids have started to emerge from the presented analyses.
Asunto(s)
Crithidia/genética , Trypanosomatina/genética , Animales , Crithidia/clasificación , Crithidia/ultraestructura , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Filogenia , Reacción en Cadena de la Polimerasa , ARN Protozoario/genética , ARN Ribosómico/genética , ARN Lider Empalmado/genética , Trypanosomatina/clasificación , Trypanosomatina/ultraestructuraRESUMEN
Trypanosomes and leishmanias are widely known parasites of humans. However, they are just two out of several phylogenetic lineages that constitute the family Trypanosomatidae. Although dixeny - the ability to infect two hosts - is a derived trait of vertebrate-infecting parasites, the majority of trypanosomatids are monoxenous. Like their common ancestor, the monoxenous Trypanosomatidae are mostly parasites or commensals of insects. This review covers recent advances in the study of insect trypanosomatids, highlighting their diversity as well as genetic, morphological and biochemical complexity, which, until recently, was underappreciated. The investigation of insect trypanosomatids is providing an important foundation for understanding the origin and evolution of parasitism, including colonization of vertebrates and the appearance of human pathogens.
Asunto(s)
Evolución Biológica , Insectos/parasitología , Trypanosomatina/clasificación , Animales , Biodiversidad , Interacciones Huésped-Parásitos , Humanos , Trypanosomatina/genética , Trypanosomatina/fisiologíaRESUMEN
The experimental approach to revealing the genetic information hidden in kinetoplastid cryptogenes and expressed through the posttranscriptional mRNA processing of U-insertion/deletion editing proceeds in reverse to the informational flow of the RNA editing process itself. While the editing integrates the informational content of maxicircle-encoded cryptogenes with that of minicircle-encoded gRNAs to produce functional edited mRNAs, the cryptogene analysis utilizes a comparison of the mature mRNA sequence with the cryptogene sequence to deduce the locations of edited sites and editing patterns, and a comparison of that mRNA sequence with the minicircle (or minicircle equivalent) sequences to identify the corresponding guide RNAs. Although a "direct" approach (prediction of a fully edited sequence pattern based on the analysis of cryptogene and minicircle sequences) seems to be theoretically possible, it proved to be not practically feasible. The major steps of the procedures utilized to decipher editing in a broad range of kinetoplastid species are presented in this chapter.
Asunto(s)
Bioquímica/métodos , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/aislamiento & purificación , Técnicas Genéticas , Genética , Kinetoplastida/genética , Edición de ARN , Trypanosoma/genética , Animales , Bisbenzimidazol/farmacología , Colorantes/farmacología , Etidio/farmacología , Sustancias Intercalantes/farmacologíaRESUMEN
A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.
Asunto(s)
Leishmania/química , Proteínas Mitocondriales/química , Proteínas Protozoarias/química , Ribonucleoproteínas/química , Animales , Microscopía por Crioelectrón , Leishmania/metabolismo , Leishmania/ultraestructura , Mitocondrias/metabolismo , Proteínas Mitocondriales/aislamiento & purificación , Proteínas Mitocondriales/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteómica , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/ultraestructura , ARN Ribosómico/química , Ribonucleoproteínas/aislamiento & purificación , Ribonucleoproteínas/ultraestructura , Proteínas Ribosómicas/química , Espectrometría de Masas en TándemRESUMEN
Mitochondrial ribosomes evolved from prokaryotic ribosomes, with which they therefore share more common features than with their counterparts in the cytosol. Yet, mitochondrial ribosomes are highly diverse in structure and composition, having undergone considerable changes, including reduction of their RNA component and varying degree of acquisition of novel proteins in various phylogenetic lineages. Here, we present functional analysis of three putative mitochondrial ribosome-associated proteins (RSM22, mtYsxC and PNKD-like) in Trypanosoma brucei, originally identified by database mining. While in other systems the homologs of RSM22 are known as components of mitochondrial ribosomes, YsxC was linked with ribosomes only in bacteria. The PNKD-like protein shows similarity to a human protein, the defects of which cause PNKD (paroxysmal non-kinesigenic dyskinesia). Here we show that all three proteins are important for the growth of T. brucei. They play an important function in mitochondrial translation, as their ablation by RNAi rapidly and severely affected the de novo synthesis of mitochondrial proteins. Moreover, following the RNAi-mediated depletion of RSM22, structure of the small subunit of mitochondrial ribosome becomes severely compromised, suggesting a role of RSM22 in ribosomal assembly and/or stability.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Biología Computacional , Silenciador del Gen , Proteínas Mitocondriales/genética , Proteínas Protozoarias/genética , Interferencia de ARN , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/genéticaRESUMEN
We have analyzed Leishmania tarentolae mitochondrial ribonucleoprotein (RNP) complexes using the 9S small subunit (SSU) rRNA and the 12S large subunit (LSU) rRNA as markers, and have identified a 50S RNP particle as the putative mitochondrial monosome, a 40S particle as the putative LSU and a 30S particle as the putative SSU. These assignments are supported by morphological analysis by cryo-electron microscopy and proteomics analyses by mass spectrometry. The presence of additional rRNA-containing particles complicated the analysis and most likely was the basis for previous difficulties in identification of these ribosomes; thus, in addition to the monosomes and their subunits, there are abundant stable 45S particles (SSU(*)) containing only 9S rRNA, which may represent homodimers of the SSU or SSU associated with additional proteins, and variable minor amounts of 65S and 70S particles, which represent homodimers of the LSU and SSU(*), respectively. These additional rRNA particles might be due to the lengthy mitochondrial isolation and ribosome isolation procedures or may be present in vivo and play yet undetermined roles.
Asunto(s)
Leishmania/citología , Mitocondrias , Subunidades de Proteína/análisis , Proteínas Protozoarias/análisis , Ribonucleoproteínas/aislamiento & purificación , Ribosomas/química , Ribosomas/ultraestructura , Animales , Microscopía por Crioelectrón , Espectrometría de Masas , Microscopía Electrónica , Peso Molecular , Subunidades de Proteína/química , Proteínas Protozoarias/química , Ribonucleoproteínas/químicaRESUMEN
The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.