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

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole-cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

• Keywords
• FISH, RNA binding protein, RNAseq, mRNA export, nucleoporin, trypanosome,
• MeSH
• Active Transport, Cell Nucleus MeSH
• Gene Knockdown Techniques methods   MeSH
• Membrane Transport Proteins metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Nucleocytoplasmic Transport Proteins metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• RNA, Transfer metabolism   MeSH
• Transcriptome MeSH
• RNA Transport MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Membrane Transport Proteins MeSH
• RNA, Messenger MeSH
• Nucleocytoplasmic Transport Proteins MeSH
• RNA-Binding Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Transfer MeSH

Transfer RNAs play a key role in protein synthesis. Following transcription, tRNAs are extensively processed prior to their departure from the nucleus to become fully functional during translation. This includes removal of 5′ leaders and 3′ trailers by a specific endo- and/or exonuclease, 3′ CCA tail addition, posttranscriptional modifications and in some cases intron removal. In this minireview, the critical factors of nuclear tRNA trafficking are described based on studies in classical models such as yeast and human cell lines. In addition, recent findings and identification of novel regulatory loops of nuclear tRNA trafficking in trypanosomes are discussed with emphasis on tRNA modifications. The comparison between the representatives of opisthokonts and excavates serves here to understand the evolutionary conservation and diversity of nuclear tRNA export mechanisms.

• Keywords
• Nuclear tRNA export, Trypanosoma brucei, tRNA modification,
• MeSH
• Cell Line MeSH
• Humans MeSH
• RNA, Nuclear genetics   metabolism   MeSH
• RNA, Transfer genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Trypanosoma genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Nuclear MeSH
• RNA, Transfer MeSH

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
• Anticodon genetics   MeSH
• Cell Nucleus genetics   ultrastructure   MeSH
• Cytoplasm genetics   ultrastructure   MeSH
• Guanosine genetics   MeSH
• Codon genetics   MeSH
• Nucleic Acid Conformation * MeSH
• Mitochondria genetics   MeSH
• Nucleoside Q genetics   MeSH
• RNA Processing, Post-Transcriptional genetics   MeSH
• Protein Biosynthesis genetics   MeSH
• RNA, Transfer genetics   ultrastructure   MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Anticodon MeSH
• Guanosine MeSH
• Codon MeSH
• Nucleoside Q MeSH
• RNA, Transfer 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 Karyopherins antagonists & inhibitors   genetics   metabolism   MeSH
• Biological Transport MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Cytoplasm genetics   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Nucleocytoplasmic Transport Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Nucleoside Q chemistry   metabolism   MeSH
• Protein Biosynthesis MeSH
• Protozoan Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• RNA, Protozoan chemistry   genetics   metabolism   MeSH
• RNA, Transfer chemistry   genetics   metabolism   MeSH
• Signal Transduction MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• beta Karyopherins MeSH
• RNA, Small Interfering MeSH
• RNA, Messenger MeSH
• Nucleocytoplasmic Transport Proteins MeSH
• Nucleoside Q MeSH
• Protozoan Proteins MeSH
• RNA, Protozoan MeSH
• RNA, Transfer MeSH