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.
- MeSH
- antikodon genetika MeSH
- buněčné jádro genetika ultrastruktura MeSH
- cytoplazma genetika ultrastruktura MeSH
- guanosin genetika MeSH
- kodon genetika MeSH
- konformace nukleové kyseliny * MeSH
- mitochondrie genetika MeSH
- nukleosid Q genetika MeSH
- posttranskripční úpravy RNA genetika MeSH
- proteosyntéza genetika MeSH
- RNA transferová genetika ultrastruktura MeSH
- Trypanosoma brucei brucei genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs. We present the iCLIP technique modified for its application to Trypanosoma brucei and most likely other kinetoplastid flagellates. By using the immuno- or affinity purification approach, it was successfully applied to the analysis of several RBPs. Furthermore, we also provide a detailed description of the iCLIP/iCLAP protocol that shall be particularly suitable for the studies of trypanosome RBPs.
- MeSH
- imunoprecipitace metody MeSH
- nukleotidy genetika metabolismus MeSH
- parazitologie metody MeSH
- proteiny vázající RNA analýza genetika metabolismus MeSH
- protozoální proteiny analýza genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- RNA genetika metabolismus MeSH
- Trypanosoma brucei brucei genetika MeSH
- ultrafialové záření MeSH
- vazba proteinů genetika účinky záření MeSH
- vazebná místa genetika MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- beta karyoferiny antagonisté a inhibitory genetika metabolismus MeSH
- biologický transport MeSH
- buněčné jádro genetika metabolismus MeSH
- cytoplazma genetika metabolismus MeSH
- konformace nukleové kyseliny MeSH
- malá interferující RNA genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- nukleocytoplazmatické transportní proteiny antagonisté a inhibitory genetika metabolismus MeSH
- nukleosid Q chemie metabolismus MeSH
- proteosyntéza MeSH
- protozoální proteiny antagonisté a inhibitory genetika metabolismus MeSH
- regulace genové exprese MeSH
- RNA protozoální chemie genetika metabolismus MeSH
- RNA transferová chemie genetika metabolismus MeSH
- signální transdukce MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
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.
- MeSH
- aktivní transport - buněčné jádro MeSH
- buněčné jádro genetika metabolismus MeSH
- cytoplazma genetika metabolismus MeSH
- kinetika MeSH
- konformace nukleové kyseliny MeSH
- nukleosid Q metabolismus MeSH
- pentosyltransferasy genetika metabolismus MeSH
- RNA transferová Phe genetika metabolismus MeSH
- RNA transferová Tyr genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sestřih RNA MeSH
- transport RNA MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
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.
- MeSH
- cytoplazma genetika MeSH
- genom mitochondriální genetika MeSH
- intracelulární membrány MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie genetika MeSH
- posttranskripční úpravy RNA genetika MeSH
- proteosyntéza genetika MeSH
- RNA transferová genetika MeSH
- symbióza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
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.
- MeSH
- esenciální geny * MeSH
- malá jadérková RNA metabolismus MeSH
- posttranskripční úpravy RNA * MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA ribozomální 18S metabolismus MeSH
- Trypanosoma brucei brucei enzymologie metabolismus MeSH
- umlčování genů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- introny * MeSH
- RNA transferová Tyr metabolismus MeSH
- Trypanosoma brucei brucei metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- guanosin analogy a deriváty biosyntéza chemie metabolismus MeSH
- mitochondrie enzymologie MeSH
- posttranskripční úpravy RNA MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA transferová chemie metabolismus MeSH
- Trypanosoma brucei brucei enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Trypanosoma brucei brucei has two distinct developmental stages, the procyclic stage in the insect and the bloodstream stage in the mammalian host. The significance of each developmental stage is punctuated by specific changes in metabolism. In the insect, T. b. brucei is strictly dependent on mitochondrial function and thus respiration to generate the bulk of its ATP, whereas in the mammalian host it relies heavily on glycolysis. These observations have raised questions about the importance of mitochondrial function in the bloodstream stage. Peculiarly, akinetoplastic strains of Trypanosoma brucei evansi that lack mitochondrial DNA do exist in the wild and are developmentally locked in the glycolysis-dependent bloodstream stage. Using RNAi we show that two mitochondrion-imported proteins, mitochondrial RNA polymerase and guide RNA associated protein 1, are still imported into the nucleic acids-lacking organelle of T. b. evansi, making the need for these proteins futile. We also show that, like in the T. b. brucei procyclic stage, the mitochondria of both bloodstream stage of T. b. brucei and T. b. evansi import various tRNAs, including those that undergo thiolation. However, we were unable to detect mitochondrial thiolation in the akinetoplastic organelle. Taken together, these data suggest a lack of connection between nuclear and mitochondrial communication in strains of T. b. evansi that lost mitochondrial genome and that do not required an insect vector for survival.
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčné jádro genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika metabolismus MeSH
- geneticky modifikované organismy MeSH
- glykolýza fyziologie MeSH
- guide RNA, Kinetoplastida metabolismus MeSH
- kinetoplastová DNA metabolismus MeSH
- mezibuněčná komunikace MeSH
- mitochondrie genetika metabolismus MeSH
- oxidativní fosforylace MeSH
- proteiny genetika metabolismus MeSH
- RNA interference MeSH
- RNA transferová genetika metabolismus MeSH
- transport proteinů MeSH
- transport RNA MeSH
- Trypanosoma fyziologie MeSH
- trypanozomiáza parazitologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
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.
- MeSH
- akonitáthydratasa metabolismus MeSH
- cytosol metabolismus MeSH
- fenotyp MeSH
- fumarasa metabolismus MeSH
- membránový potenciál mitochondrií MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- proteiny obsahující železo a síru metabolismus MeSH
- protozoální proteiny metabolismus MeSH
- RNA interference MeSH
- RNA protozoální metabolismus MeSH
- RNA transferová metabolismus MeSH
- stabilita proteinů MeSH
- sulfhydrylové sloučeniny metabolismus MeSH
- Trypanosoma brucei brucei cytologie enzymologie růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH