RESUMO
The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.
Assuntos
Núcleo Celular , Genoma Mitocondrial , Edição de RNA , RNA de Transferência , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trypanosomatina/genética , Trypanosomatina/metabolismo , Códon/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Códon de Terminação/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Código Genético , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.
Assuntos
Anticódon , Códon de Terminação , Células Eucarióticas , Código Genético , Mutação , Fatores de Terminação de Peptídeos , RNA de Transferência , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Cilióforos/genética , Códon de Terminação/genética , Código Genético/genética , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Triptofano/genética , Saccharomyces cerevisiae/genética , RNA de Transferência de Ácido Glutâmico/genética , Trypanosoma brucei brucei/genéticaRESUMO
The nuclear pore complex (NPC) facilitates the trafficking of proteins and RNA between the nucleus and cytoplasm. The role of nucleoporins (Nups) in transport in the context of the NPC is well established, yet their function in tRNA export has not been fully explored. We selected several nucleoporins from different parts of the NPC to investigate their potential role in tRNA trafficking in Trypanosoma brucei. We show that while all of the nucleoporins studied are essential for cell viability, only TbNup62 and TbNup53a function in tRNA export. In contrast to homologs in yeast TbNup144 and TbNup158, which are part of the inner and outer ring of the NPC, have no role in nuclear tRNA trafficking. Instead, TbNup144 plays a critical role in nuclear division, highlighting the role of nucleoporins beyond nucleocytoplasmic transport. These results suggest that the location of nucleoporins within the NPC is crucial to maintaining various cellular processes.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.
Assuntos
Códon/metabolismo , Nucleosídeo Q/metabolismo , Nutrientes/metabolismo , RNA de Transferência/metabolismo , Trypanosoma brucei brucei/metabolismo , Aminoácidos/metabolismo , Cromatografia Líquida/métodos , Códon/genética , Guanina/análogos & derivados , Guanina/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Splicing de RNA , RNA de Transferência/genética , RNA de Transferência de Tirosina/genética , RNA de Transferência de Tirosina/metabolismo , Espectrometria de Massas em Tandem/métodos , Trypanosoma brucei brucei/genética , Tirosina/metabolismoRESUMO
Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.
Assuntos
Mitocôndrias/genética , Conformação de Ácido Nucleico , Nucleosídeo Q/genética , RNA de Transferência/genética , Anticódon/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Códon/genética , Citoplasma/genética , Citoplasma/ultraestrutura , Guanosina/genética , Biossíntese de Proteínas/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/ultraestrutura , Trypanosoma brucei brucei/genéticaRESUMO
In kinetoplastid protists, all mitochondrial tRNAs are encoded in the nucleus and imported from the cytoplasm to maintain organellar translation. This also applies to the tryptophanyl tRNA (tRNATrp) encoded by a single-copy nuclear gene, with a CCA anticodon to read UGG codon used in the cytosolic translation. Yet, in the mitochondrion it is unable to decode the UGA codon specifying tryptophan. Following mitochondrial import of tRNATrp, this problem is solved at the RNA level by a single C34 to U34 editing event that creates the UCA anticodon, recognizing UGA. To identify the enzyme responsible for this critical editing activity, we scrutinized the genome of Trypanosoma brucei for putative cytidine deaminases as the most likely candidates. Using RNAi silencing and poisoned primer extension, we have identified a novel deaminase enzyme, named here TbmCDAT for mitochondrial Cytidine Deaminase Acting on tRNA, which is responsible for this organelle-specific activity in T. brucei. The ablation of TbmCDAT led to the downregulation of mitochondrial protein synthesis, supporting its role in decoding the UGA tryptophan codon.
Assuntos
Códon de Terminação , Citidina Desaminase/metabolismo , Citidina/genética , Mitocôndrias/enzimologia , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Uridina/genética , Sequência de Aminoácidos , Sequência de Bases , Citidina/química , Citidina Desaminase/genética , Mitocôndrias/genética , Conformação de Ácido Nucleico , RNA Mitocondrial/análise , RNA Mitocondrial/genética , RNA de Protozoário/análise , RNA de Transferência de Triptofano , Homologia de Sequência , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Uridina/químicaRESUMO
Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole-cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.
Assuntos
Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Transporte Ativo do Núcleo Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Transporte de RNA , RNA de Transferência/metabolismo , TranscriptomaRESUMO
Transfer RNAs play a key role in protein synthesis. Following transcription, tRNAs are extensively processed prior to their departure from the nucleus to become fully functional during translation. This includes removal of 5' leaders and 3' trailers by a specific endo- and/or exonuclease, 3' CCA tail addition, posttranscriptional modifications and in some cases intron removal. In this minireview, the critical factors of nuclear tRNA trafficking are described based on studies in classical models such as yeast and human cell lines. In addition, recent findings and identification of novel regulatory loops of nuclear tRNA trafficking in trypanosomes are discussed with emphasis on tRNA modifications. The comparison between the representatives of opisthokonts and excavates serves here to understand the evolutionary conservation and diversity of nuclear tRNA export mechanisms.
Assuntos
RNA Nuclear/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Trypanosoma/metabolismo , Linhagem Celular , Humanos , RNA Nuclear/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Trypanosoma/genéticaRESUMO
Transfer RNAs (tRNAs) are central players in protein synthesis, which in Eukarya need to be delivered from the nucleus to the cytoplasm by specific transport receptors, most of which belong to the evolutionarily conserved beta-importin family. Based on the available literature, we identified two candidates, Xpo-t and Xpo-5 for tRNA export in Trypanosoma brucei. However, down-regulation of expression of these genes did not disrupt the export of tRNAs to the cytoplasm. In search of alternative pathways, we tested the mRNA export complex Mex67-Mtr2, for a role in tRNA nuclear export, as described previously in yeast. Down-regulation of either exporter affected the subcellular distribution of tRNAs. However, contrary to yeast, TbMex67 and TbMtr2 accumulated different subsets of tRNAs in the nucleus. While TbMtr2 perturbed the export of all the tRNAs tested, silencing of TbMex67, led to the nuclear accumulation of tRNAs that are typically modified with queuosine. In turn, inhibition of tRNA nuclear export also affected the levels of queuosine modification in tRNAs. Taken together, the results presented demonstrate the dynamic nature of tRNA trafficking in T. brucei and its potential impact not only on the availability of tRNAs for protein synthesis but also on their modification status.
Assuntos
Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA de Transferência/genética , Trypanosoma brucei brucei/genética , beta Carioferinas/genética , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inibidores , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Nucleosídeo Q/química , Nucleosídeo Q/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/química , RNA de Protozoário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Transdução de Sinais , Trypanosoma brucei brucei/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/metabolismoRESUMO
Organisms have evolved different strategies to seclude certain molecules to specific locations of the cell. This is most pronounced in eukaryotes with their extensive intracellular membrane systems. Intracellular compartmentalization is particularly critical in genome containing organelles, which because of their bacterial evolutionary ancestry still maintain protein-synthesis machinery that resembles more their evolutionary origin than the extant eukaryotic cell they once joined as an endosymbiont. Despite this, it is clear that genome-containing organelles such as the mitochondria are not in isolation and many molecules make it across the mitochondrial membranes from the cytoplasm. In this realm the import of tRNAs and the enzymes that modify them prove most consequential. In this review, we discuss two recent examples of how modifications typically found in cytoplasmic tRNAs affect mitochondrial translation in organisms that forcibly import all their tRNAs from the cytoplasm. In our view, the combination of tRNA import and the compartmentalization of modification enzymes must have played a critical role in the evolution of the organelle. © 2018 IUBMB Life, 70(12):1207-1213, 2018.
Assuntos
Genoma Mitocondrial/genética , Mitocôndrias/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Citoplasma/genética , Membranas Intracelulares , Membranas Mitocondriais/metabolismo , Processamento Pós-Transcricional do RNA/genética , Simbiose/genéticaRESUMO
Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Pentosiltransferases/genética , RNA de Transferência de Tirosina/genética , Trypanosoma brucei brucei/genética , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Citoplasma/genética , Cinética , Conformação de Ácido Nucleico , Nucleosídeo Q/metabolismo , Pentosiltransferases/metabolismo , Splicing de RNA , Transporte de RNA , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/metabolismo , RNA de Transferência de Tirosina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes 'wholesale deamination' of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.
Assuntos
Metiltransferases/metabolismo , Nucleosídeo Desaminases/metabolismo , Edição de RNA , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Anticódon/metabolismo , Sequência de Bases , Citosina/análogos & derivados , Citosina/metabolismo , Desaminação , Metilação , RNA de Transferência de Treonina/genética , Uridina/metabolismoRESUMO
Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA(Tyr), and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA(Tyr) and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an "intronless" version of tRNA(Tyr) in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA(Tyr) maturation and viability, while revealing tRNA splicing as its only essential function.
Assuntos
Íntrons , RNA de Transferência de Tirosina/metabolismo , Trypanosoma brucei brucei/metabolismo , AnimaisRESUMO
Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells.
Assuntos
Divisão Celular/fisiologia , Metiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Ribossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Humanos , Metiltransferases/genética , Proteínas de Protozoários/genética , Ribossomos/genética , Trypanosoma brucei brucei/genéticaRESUMO
In this review the main features of the mitochondria of aerobic parasitic protists are discussed. While the best characterized organelles are by far those of kinetoplastid flagellates and Plasmodium, we also consider amoebae Naegleria and Acanthamoeba, a ciliate Ichthyophthirius and related lineages. The simplistic view of the mitochondrion as just a power house of the cell has already been abandoned in multicellular organisms and available data indicate that this also does not apply for protists. We discuss in more details the following mitochondrial features: genomes, post-transcriptional processing, translation, biogenesis of iron-sulfur complexes, heme metabolism and the electron transport chain. Substantial differences in all these core mitochondrial features between lineages are compatible with the view that aerobic protists harbor organelles that are more complex and flexible than previously appreciated.
Assuntos
Genoma Microbiano , Mitocôndrias/genética , Mitocôndrias/metabolismo , Parasitos/genética , Parasitos/metabolismo , Animais , Replicação do DNA , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Variação Estrutural do Genoma , Heme/metabolismo , Ferro/metabolismo , Ribossomos Mitocondriais/metabolismo , Oxigênio/metabolismo , RNA/genética , RNA/metabolismo , Edição de RNA , RNA Mitocondrial , Enxofre/metabolismoRESUMO
ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Aconitato Hidratase/metabolismo , Citosol/metabolismo , Fumarato Hidratase/metabolismo , Técnicas de Silenciamento de Genes , Genes de Protozoários , Teste de Complementação Genética , Heme/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Filogenia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/genéticaRESUMO
Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.