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
• Nucleoside Q metabolism   analogs & derivatives   MeSH
• RNA Processing, Post-Transcriptional * MeSH
• Protein Domains MeSH
• RNA-Binding Proteins * metabolism   chemistry   genetics   MeSH
• Protozoan Proteins * metabolism   chemistry   genetics   MeSH
• RNA, Transfer * metabolism   genetics   MeSH
• Trypanosoma brucei brucei * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nucleoside Q MeSH
• RNA-Binding Proteins * MeSH
• Protozoan Proteins * MeSH
• RNA, Transfer * 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
• Amino Acids metabolism   MeSH
• Chromatography, Liquid methods   MeSH
• Guanine analogs & derivatives   metabolism   MeSH
• Codon genetics   metabolism   MeSH
• Nucleoside Q metabolism   MeSH
• Pentosyltransferases genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA, Transfer, Tyr genetics   metabolism   MeSH
• RNA, Transfer genetics   metabolism   MeSH
• RNA Splicing MeSH
• Tandem Mass Spectrometry methods   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Tyrosine metabolism   MeSH
• Nutrients metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Amino Acids MeSH
• Guanine MeSH
• Codon MeSH
• Nucleoside Q MeSH
• Pentosyltransferases MeSH
• Protozoan Proteins MeSH
• queuine tRNA-ribosyltransferase MeSH Browser
• queuine MeSH Browser
• RNA, Transfer, Tyr MeSH
• RNA, Transfer MeSH
• Tyrosine MeSH