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.

• Klíčová slova
• FG-Nups, NPC, Trypanosoma brucei, nuclear division, nucleoporins, tRNA trafficking,
• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• jaderný pór * genetika   metabolismus   MeSH
• komplex proteinů jaderného póru * genetika   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• komplex proteinů jaderného póru * MeSH

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.

• MeSH
• aminokyseliny metabolismus   MeSH
• chromatografie kapalinová metody   MeSH
• guanin analogy a deriváty   metabolismus   MeSH
• kodon genetika   metabolismus   MeSH
• nukleosid Q metabolismus   MeSH
• pentosyltransferasy genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA transferová Tyr genetika   metabolismus   MeSH
• RNA transferová genetika   metabolismus   MeSH
• sestřih RNA MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• živiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aminokyseliny MeSH
• guanin MeSH
• kodon MeSH
• nukleosid Q MeSH
• pentosyltransferasy MeSH
• protozoální proteiny MeSH
• queuine tRNA-ribosyltransferase MeSH Prohlížeč
• queuine MeSH Prohlížeč
• RNA transferová Tyr MeSH
• RNA transferová MeSH
• tyrosin 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
• Názvy látek
• beta karyoferiny MeSH
• malá interferující RNA MeSH
• messenger RNA MeSH
• nukleocytoplazmatické transportní proteiny MeSH
• nukleosid Q MeSH
• protozoální proteiny MeSH
• RNA protozoální MeSH
• RNA transferová 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.

• Klíčová slova
• 1-methylguanosine, import, mitochondria, trypanosomes, wybutosine,
• 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
• Názvy látek
• RNA transferová 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.

• Klíčová slova
• Intron, queuosine, retrograde, splicing, tRNA, transport,
• 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
• Názvy látek
• nukleosid Q MeSH
• pentosyltransferasy MeSH
• queuine tRNA-ribosyltransferase MeSH Prohlížeč
• RNA transferová Phe MeSH
• RNA transferová Tyr MeSH