The RNA-binding protein La is found in most eukaryotes, and despite being essential in many organisms, its function is not completely clear. Trypanosoma brucei, the causative agent of human African trypanosomiasis, encodes a 'classical' La protein (TbLa) composed of a La-motif, two RNA recognition motifs (RRM1 and RRM2α), a C-terminal short basic motif (SBM), and a nuclear localization signal (NLS). In T. brucei, like in most eukaryotes, position 34 of tRNATyr, -Asp, -Asn and -His is modified with queuosine (Q34). The steady-state levels of queuosine-modified tRNA in the insect form (procyclic) of T. brucei can fluctuate dynamically depending on growth conditions, but the mechanism(s) controlling Q34 levels are not well understood. A well-established function of La is in precursor-tRNA 3'-end metabolism, but in this work, we demonstrate that La also controls Q34-tRNA levels. Individual domain deletions showed that while deletion of La motif or RRM1 causes dysregulation of Q34-tRNA levels, no other domain plays a similar role. We also show that La is important for the normal balance of several additional tRNA modifications. These findings are discussed in the context of substrate competition between La and modification enzymes, also highlighting subcellular localization as a key determinant of tRNA function.

• MeSH
• nukleosid Q metabolismus   analogy a deriváty   MeSH
• posttranskripční úpravy RNA * MeSH
• proteinové domény MeSH
• proteiny vázající RNA * metabolismus   chemie   genetika   MeSH
• protozoální proteiny * metabolismus   chemie   genetika   MeSH
• RNA transferová * metabolismus   genetika   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nukleosid Q MeSH
• proteiny vázající RNA * MeSH
• protozoální proteiny * 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

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.

• MeSH
• antikodon metabolismus   MeSH
• cytosin analogy a deriváty   metabolismus   MeSH
• deaminace MeSH
• editace RNA * MeSH
• methyltransferasy metabolismus   MeSH
• metylace MeSH
• nukleosiddeaminasy metabolismus   MeSH
• RNA transferová Thr chemie   genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Trypanosoma brucei brucei enzymologie   genetika   MeSH
• uridin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• 3-methylcytosine MeSH Prohlížeč
• antikodon MeSH
• cytosin MeSH
• methyltransferasy MeSH
• nukleosiddeaminasy MeSH
• RNA transferová Thr MeSH
• uridin MeSH