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