ABSTRACT
The essential deamination of adenosine A34 to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A34 deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.
Subject(s)
Adenosine Deaminase , Eukaryota , Adenosine Deaminase/metabolism , Eukaryota/genetics , Eukaryota/metabolism , Inosine/metabolism , RNA, Transfer/metabolism , Anticodon/geneticsABSTRACT
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.
Subject(s)
Codon/metabolism , Nucleoside Q/metabolism , Nutrients/metabolism , RNA, Transfer/metabolism , Trypanosoma brucei brucei/metabolism , Amino Acids/metabolism , Chromatography, Liquid/methods , Codon/genetics , Guanine/analogs & derivatives , Guanine/metabolism , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA Splicing , RNA, Transfer/genetics , RNA, Transfer, Tyr/genetics , RNA, Transfer, Tyr/metabolism , Tandem Mass Spectrometry/methods , Trypanosoma brucei brucei/genetics , Tyrosine/metabolismABSTRACT
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.
Subject(s)
Mitochondria/genetics , Nucleic Acid Conformation , Nucleoside Q/genetics , RNA, Transfer/genetics , Anticodon/genetics , Cell Nucleus/genetics , Cell Nucleus/ultrastructure , Codon/genetics , Cytoplasm/genetics , Cytoplasm/ultrastructure , Guanosine/genetics , Protein Biosynthesis/genetics , RNA Processing, Post-Transcriptional/genetics , RNA, Transfer/ultrastructure , Trypanosoma brucei brucei/geneticsABSTRACT
Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.
Subject(s)
Genes, Essential , Protozoan Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA, Ribosomal, 18S/metabolism , RNA, Small Nucleolar/metabolism , RNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/enzymology , Gene Silencing , Protozoan Proteins/genetics , RNA-Binding Proteins/genetics , Trypanosoma brucei brucei/metabolismABSTRACT
Transfer RNAs acquire a variety of naturally occurring chemical modifications during their maturation; these fine-tune their structure and decoding properties in a manner critical for protein synthesis. We recently reported that in the eukaryotic parasite, Trypanosoma brucei, a methylation and deamination event are unexpectedly interconnected, whereby the tRNA adenosine deaminase (TbADAT2/3) and the 3-methylcytosine methyltransferase (TbTrm140) strictly rely on each other for activity, leading to formation of m3C and m3U at position 32 in several tRNAs. Still however, it is not clear why these two enzymes, which work independently in other systems, are strictly codependent in T. brucei Here, we show that these enzymes exhibit binding synergism, or a mutual increase in binding affinity, that is more than the sum of the parts, when added together in a reaction. Although these enzymes interact directly with each other, tRNA binding assays using enzyme variants mutated in critical binding and catalytic sites indicate that the observed binding synergy stems from contributions from tRNA-binding domains distal to their active sites. These results provide a rationale for the known interactions of these proteins, while also speaking to the modulation of substrate specificity between seemingly unrelated enzymes. This information should be of value in furthering our understanding of how tRNA modification enzymes act together to regulate gene expression at the post-transcriptional level and provide a basis for the interdependence of such activities.
Subject(s)
Protozoan Proteins/chemistry , RNA, Protozoan/chemistry , RNA, Transfer/chemistry , RNA-Binding Proteins/chemistry , Trypanosoma brucei brucei/enzymology , Kinetics , Protein Binding , Protozoan Proteins/physiology , RNA Editing , RNA-Binding Proteins/physiology , ThermodynamicsABSTRACT
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.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Pentosyltransferases/genetics , RNA, Transfer, Tyr/genetics , Trypanosoma brucei brucei/genetics , Active Transport, Cell Nucleus , Cell Nucleus/genetics , Cytoplasm/genetics , Kinetics , Nucleic Acid Conformation , Nucleoside Q/metabolism , Pentosyltransferases/metabolism , RNA Splicing , RNA Transport , RNA, Transfer, Phe/genetics , RNA, Transfer, Phe/metabolism , RNA, Transfer, Tyr/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Trypanosoma brucei brucei/metabolismABSTRACT
All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. 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 [1]. In tRNA, the function of modifications varies; some modulate global and/or local RNA structure, and others directly impact decoding and may be essential for viability. Whichever the case, the overall importance of modifications is highlighted by both their evolutionary conservation and the fact that organisms use a substantial portion of their genomes to encode modification enzymes, far exceeding what is needed for the de novo synthesis of the canonical nucleotides themselves [2]. Although some modifications occur at exactly the same nucleotide position in tRNAs from the three domains of life, many can be found at various positions in a particular tRNA and their location may vary between and within different tRNAs. With this wild array of chemical diversity and substrate specificities, one of the big challenges in the tRNA modification field has been to better understand at a molecular level the modes of substrate recognition by the different modification enzymes; in this realm RNA binding rests at the heart of the problem. This chapter will focus on several examples of modification enzymes where their mode of RNA binding is well understood; from these, we will try to draw general conclusions and highlight growing themes that may be applicable to the RNA modification field at large.
Subject(s)
Enzymes/metabolism , Evolution, Molecular , RNA Processing, Post-Transcriptional , RNA, Transfer/chemistry , RNA, Transfer/metabolism , RNA, Transfer/genetics , Substrate SpecificityABSTRACT
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.
Subject(s)
Methyltransferases/metabolism , Nucleoside Deaminases/metabolism , RNA Editing , RNA, Transfer, Thr/chemistry , RNA, Transfer, Thr/metabolism , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/genetics , Anticodon/metabolism , Base Sequence , Cytosine/analogs & derivatives , Cytosine/metabolism , Deamination , Methylation , RNA, Transfer, Thr/genetics , Uridine/metabolismABSTRACT
Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are modifications found at position 37, 3Î-adjacent to the anticodon, in tRNAs responsible for ANN codons. These modifications are universally conserved in all domains of life. t6A and its derivatives have pleiotropic functions in protein synthesis including aminoacylation, decoding and translocation. We previously discovered a cyclic form of t6A (ct6A) as a chemically labile derivative of t6A in tRNAs from bacteria, fungi, plants and protists. Here, we report 2-methylthio cyclic t6A (ms2ct6A), a novel derivative of ct6A found in tRNAs from Bacillus subtilis, plants and Trypanosoma brucei. In B. subtilis and T. brucei, ms2ct6A disappeared and remained to be ms2t6A and ct6A by depletion of tcdA and mtaB homologs, respectively, demonstrating that TcdA and MtaB are responsible for biogenesis of ms2ct6A.
Subject(s)
Adenosine/analogs & derivatives , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Threonine/analogs & derivatives , Adenosine/chemistry , Adenosine/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Base Sequence , Nucleic Acid Conformation , Phenotype , RNA, Plant/chemistry , RNA, Plant/metabolism , Spectrometry, Mass, Electrospray Ionization , Threonine/chemistry , Threonine/metabolismABSTRACT
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.
Subject(s)
Cell Division/physiology , Methyltransferases/metabolism , Protozoan Proteins/metabolism , Ribosomes/metabolism , Trypanosoma brucei brucei/metabolism , Humans , Methyltransferases/genetics , Protozoan Proteins/genetics , Ribosomes/genetics , Trypanosoma brucei brucei/geneticsABSTRACT
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.
Subject(s)
Guanosine/analogs & derivatives , Mitochondria/enzymology , RNA, Transfer/metabolism , Trypanosoma brucei brucei/enzymology , Guanosine/biosynthesis , Guanosine/chemistry , Guanosine/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA, Transfer/chemistry , Trypanosoma brucei brucei/geneticsABSTRACT
In cells, tRNAs are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends. Some tRNAs also contain introns, which, in archaea and eukaryotes, are cleaved by an evolutionarily conserved endonuclease complex that generates fully functional mature tRNAs. In addition, tRNAs undergo numerous posttranscriptional nucleotide chemical modifications. In Trypanosoma brucei, the single intron-containing tRNA (tRNA(Tyr)GUA) is responsible for decoding all tyrosine codons; therefore, intron removal is essential for viability. Using molecular and biochemical approaches, we show the presence of several noncanonical editing events, within the intron of pre-tRNA(Tyr)GUA, involving guanosine-to-adenosine transitions (G to A) and an adenosine-to-uridine transversion (A to U). The RNA editing described here is required for proper processing of the intron, establishing the functional significance of noncanonical editing with implications for tRNA processing in the deeply divergent kinetoplastid lineage and eukaryotes in general.
Subject(s)
Introns/genetics , RNA Editing , RNA Splicing , RNA, Transfer, Tyr/genetics , Trypanosoma brucei brucei/genetics , Amino Acid Sequence , Base Sequence , Blotting, Northern , Endoribonucleases/genetics , Endoribonucleases/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA Interference , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , RNA, Transfer, Tyr/chemistry , RNA, Transfer, Tyr/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Trypanosoma brucei brucei/metabolismABSTRACT
All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions in translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m(1)G37) of the anticodon loop in several tRNAs, is evolutionarily conserved and participates in translational reading frame maintenance. In eukaryotes, the tRNA methyltransferase TRM5 is responsible for m(1)G formation in nucleus-encoded as well as mitochondria-encoded tRNAs, reflecting the universal importance of this modification for protein synthesis. However, it is not clear what role, if any, mitochondrial TRM5 serves in organisms that do not encode tRNAs in their mitochondrial genomes. These organisms may easily satisfy the m(1)G37 requirement through their robust mitochondrial tRNA import mechanisms. We have explored this possibility in the parasitic protist Trypanosoma brucei and show that down-regulation of TRM5 by RNAi leads to the expected disappearance of m(1)G37, but with surprisingly little effect on cytoplasmic translation. On the contrary, lack of TRM5 causes a marked growth phenotype and a significant decrease in mitochondrial functions, including protein synthesis. These results suggest mitochondrial TRM5 may be needed to mature unmethylated tRNAs that reach the mitochondria and that could pose a problem for translational fidelity. This study also reveals an unexpected lack of import specificity between some fully matured and potentially defective tRNA species.
Subject(s)
Methyltransferases , Mitochondrial Proteins , RNA, Transfer , Trypanosoma brucei brucei , Anticodon/chemistry , Down-Regulation , Genome, Mitochondrial , Guanosine/analogs & derivatives , Guanosine/genetics , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Mitochondrial Proteins/biosynthesis , Mitochondrial Proteins/metabolism , Protein Biosynthesis , RNA, Transfer/chemistry , RNA, Transfer/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolismABSTRACT
Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitochondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles.
Subject(s)
RNA, Transfer/metabolism , Active Transport, Cell Nucleus , Animals , Biological Transport, Active , Cytoplasm/metabolism , Humans , Mitochondria/metabolism , Models, Biological , Models, Molecular , Nucleic Acid Conformation , Organelles/metabolism , Plants/metabolism , RNA, Fungal/metabolism , RNA, Plant/metabolism , RNA, Protozoan/metabolism , RNA, Transfer/chemistry , Saccharomyces cerevisiae/metabolism , Trypanosomatina/metabolismABSTRACT
Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.
Subject(s)
Adenosine Deaminase/metabolism , Protozoan Proteins/metabolism , RNA Editing/physiology , RNA, Protozoan/biosynthesis , RNA, Transfer/biosynthesis , Trypanosoma brucei brucei/enzymology , Zinc/metabolism , Adenosine Deaminase/genetics , Cations, Divalent/metabolism , Protozoan Proteins/genetics , RNA, Protozoan/genetics , RNA, Transfer/genetics , RNA-Binding Proteins , Trypanosoma brucei brucei/geneticsABSTRACT
Fe/S clusters are part of the active site of many enzymes and are essential for cell viability. In eukaryotes the cysteine desulfurase Nfs (IscS) donates the sulfur during Fe/S cluster assembly and was thought sufficient for this reaction. Moreover, Nfs is indispensable for tRNA thiolation, a modification generally required for tRNA function and protein synthesis. Recently, Isd11 was discovered as an integral part of the Nfs activity at an early step of Fe/S cluster assembly. Here we show, using a combination of genetic, molecular, and biochemical approaches, that Isd11, in line with its strong association with Nfs, is localized in the mitochondrion of T. brucei. In addition to its involvement in Fe/S assembly, Isd11 also partakes in both cytoplasmic and mitochondrial tRNA thiolation, whereas Mtu1, another protein proposed to collaborate with Nfs in tRNA thiolation, is required for this process solely within the mitochondrion. Taken together these data place Isd11 at the center of these sulfur transactions and raises the possibility of a connection between Fe/S metabolism and protein synthesis, helping integrate two seemingly unrelated pathways.
Subject(s)
Iron-Sulfur Proteins/metabolism , Protozoan Proteins/metabolism , RNA, Protozoan/metabolism , RNA, Transfer/metabolism , Sulfhydryl Compounds/metabolism , Trypanosoma brucei brucei/metabolism , Aconitate Hydratase/metabolism , Cytosol/metabolism , Fumarate Hydratase/metabolism , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Phenotype , Protein Stability , RNA Interference , Trypanosoma brucei brucei/cytology , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/growth & developmentABSTRACT
Kinetoplastids encode a single nuclear tryptophanyl tRNA that contains a CCA anticodon able to decode the UGG codons used in cytoplasmic protein synthesis but cannot decode the mitochondrial UGA codons. Following mitochondrial import, this problem is circumvented in Trypanosoma brucei by specifically editing the tRNA(Trp) anticodon to UCA, which can now decode the predominant mitochondrial UGA tryptophan codons. This tRNA also undergoes an unusual thiolation at position 33 of the anticodon loop, the only known modification at U33 in any tRNA. In other organisms, tRNA thiolation is mediated by the cysteine desulfurase, Nfs1 (IscS). However, T. brucei encodes two Nfs homologues, one cytoplasmic and the other mitochondrial. We show by a combination of RNA interference and Northern and Western analyses that the mitochondria-targeted TbNfs, and not TbNfs-like protein, is essential for thiolation of both cytosolic and mitochondrial tRNAs. Given the exclusive mitochondrial localization of TbNfs, how it mediates thiolation in the cytoplasm remains unclear. Furthermore, thiolation specifically affects thiolated tRNA stability in the cytoplasm but more surprisingly acts as a negative determinant for the essential C to U editing in T. brucei. This provides a first line of evidence for mitochondrial C to U editing regulation in this system.
Subject(s)
RNA Editing/physiology , RNA Stability/physiology , RNA, Protozoan/metabolism , RNA, Transfer, Trp/metabolism , RNA/metabolism , Trypanosoma brucei brucei/metabolism , Animals , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA/genetics , RNA, Mitochondrial , RNA, Protozoan/genetics , RNA, Transfer, Trp/genetics , Trypanosoma brucei brucei/geneticsABSTRACT
Due to a complete lack of the tRNA genes in the mitochondrial genome of Trypanosoma brucei, all tRNAs needed for mitochondrial translation have to be imported into the organelle from the cytosol. A previous study showed that the modified nucleotide s(2)U could act as a negative determinant for mitochondrial tRNA import in another kinetoplastid, Leishmania tarentolae. We have investigated whether the same type of cytosolic control for tRNA retention exists in T. brucei. Based on Northern analysis with subcellular RNA fractions and in vitro import assays, we demonstrate that silencing of the cysteine desulfurase, TbNfs (TbIscS), the key enzyme in tRNA thiolation (s(2)U) and Fe-S cluster formation in vivo, has no effect on tRNA partitioning. This observation is especially surprising in light of a recent report suggesting that in L. tropica the Rieske Fe-S protein is an essential component of the RNA import complex (RIC). In line with the above observation, we also show that down-regulation of the Rieske protein by RNA interference, similar to the TbNfs knockdowns, has no effect on import. The data presented here supports the view that in T. brucei: (1) s(2)U is not a negative determinant for tRNA import; (2) the Rieske protein is not an essential component of the import machinery, and (3) since the Rieske protein is essential for respiration and maintenance of inner mitochondrial membrane potential, neither process plays a critical role in tRNA import. We therefore suggest that the T. brucei import machinery differs substantially from what has been described in Leishmania.
Subject(s)
Carbon-Sulfur Lyases/metabolism , Electron Transport Complex III/metabolism , Mitochondria/metabolism , RNA, Protozoan/metabolism , RNA, Transfer/metabolism , Sulfur/metabolism , Trypanosoma brucei brucei/metabolism , Animals , Blotting, Northern , Carbon-Sulfur Lyases/antagonists & inhibitors , Carbon-Sulfur Lyases/genetics , Cytosol/metabolism , Immunoblotting , Mitochondria/genetics , RNA, Protozoan/genetics , RNA, Small Interfering/pharmacology , RNA, Transfer/genetics , Subcellular Fractions , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & developmentABSTRACT
Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA(Gln) into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA(CUG)(Gln) and tRNA(UUG)(Gln) species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA(Gln) import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA(Gln) species and a bacterial pre-tRNA(Asp) can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.
Subject(s)
Mammals/metabolism , Mitochondria/metabolism , RNA Transport , RNA, Transfer, Gln/metabolism , Adenosine Triphosphate/pharmacology , Animals , Base Sequence , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cytosol/drug effects , Cytosol/metabolism , HeLa Cells , Humans , MERRF Syndrome/pathology , Mitochondria/drug effects , Mitochondria/pathology , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Molecular Sequence Data , Nucleic Acid Conformation/drug effects , Protein Transport/drug effects , RNA Transport/drug effects , RNA, Transfer, Gln/chemistry , RNA, Transfer, Gln/genetics , Rats , Solubility/drug effectsABSTRACT
Transfer RNA (tRNA) plays a pivotal role in protein synthesis within cells, where it is recognized by one cognate aminoacyl-tRNA synthetase, in competition with the remaining non-cognate synthetases, and esterified with an amino acid. For many years the levels of tRNA aminoacylation, in a given population of cellular RNA, have been analyzed using methods that include northern analysis and/or oxidation techniques to separate aminoacylated from non-aminoacylated species. In the present report we describe an approach recently developed by us that combines oxidation-protection with polyadenylation and PCR. The OXOPAP approach permits the amplification of tRNA species that are nearly identical and that evade differential identification by more classical northern hybridization methods. Our approach also allows the identification of aminoacylatable "naïve" species, where no prior knowledge of sequence content is necessary for amplification.