Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei

Abstract

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C-->U RNA editing in the anticodon of the imported tRNA(Trp). In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA(Trp) act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA(Trp). Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.

Fig. 1.

T. brucei contains two eukaryotic TrpRSs. (A) Multiple sequence alignment of the cytosolic TrpRS from S. cerevisiae (ScWRS1) and the two T. brucei orthologues (Tb-TrpRS1 and Tb-TrpRS2). The sequences were aligned by using the clustal w program with default parameters. Strictly conserved residues and conservative replacements are shown in black and gray boxes, respectively. (B) Position of the two trypanosomal enzymes Tb-TrpRS1 (Tb1) and Tb-TrpRS2 (Tb2) in a phylogenetic tree based on a multiple sequence alignment of the cytosolic TrpRS from mouse [Mus musculus (Mm)], Drosophila melanogaster (Dm), and yeast [S. cerevisiae (Sc)]; the mitochondrial enzyme of yeast (Sc mito) and mouse (Mm mito); and, indicated by gray letters, the TrpRS from the bacteria Corynebacterium glutamicum (Cg), Bacillus subtilis (Bs), and E. coli (Ec). The tree was constructed by using treeview, which is available on http://taxonomy.zoology.gla.ac.uk/rod/treeview.html.

Fig. 2.

Localization of trypanosomal TrpRSs. (A Top and Center) Double immunofluorescence analysis of a T. brucei cell line expressing Tb-TrpRS1 carrying the Ty1 tag at its carboxyl terminus under the control of the tetracycline-inducible (+Tet and −Tet) procyclin promoter. The cells were stained for DNA by using DAPI, for a subunit of the ATPase, serving as a mitochondrial marker and with a monoclonal antibody recognizing the Ty1-Tag. (A Bottom) Same as Top and Center, but a cell line expressing carboxyl-terminally Ty1-tagged Tb-TrpRS2 was analyzed. (Scale bars: 10 μm.) (B) Immunoblot analysis of total cellular (T), crude cytosolic (C), and crude mitochondrial extracts (M) for the presence of the Ty1-tagged Tb-TrpRS1 and Tb-TrpRS2, respectively. Elongation factor 1a (EF-1a) served as a cytosolic and α-ketoglutarate dehydrogenase (KDH) as a mitochondrial marker.

Fig. 3.

Tb-TrpRS1 and Tb-TrpRS2 are essential for the growth of procyclic T. brucei and are responsible for formation of tryptophanyl-tRNA in the cytosol and the mitochondria, respectively (A) Growth curve in the presence and absence of tetracycline (+Tet and −Tet) of a representative clonal T. brucei RNAi cell line ablated for Tb-TrpRS1. (A Inset) A Northern blot for Tb-TrpRS1 mRNA. The time of sampling is indicated by the arrow. The rRNAs in the lower panel serve as loading controls. (B) Same as A for an RNAi cell line ablated for Tb-TrpRS2. (C) Northern blot analysis of total and mitochondrial RNA isolated under acidic conditions from the Tb-TrpRS1 RNAi cell line. The total RNA fraction only contains ≈5% of mitochondrial RNA and, thus, essentially represents cytosolic RNA. Days of induction (0 and 3) by tetracycline are indicated at the bottom. The blots were probed for the T. brucei tRNA^Trp as well as tRNA^Leu and tRNA^Tyr, which serve as controls not affected by the RNAi. The RNA fractions were resolved on long acid urea gels, which allow separation of aminoacylated from deacylated tRNAs. The bar graph shows the quantification of the results. Relative amounts of aminoacylated tRNAs are indicated for the tRNA^Trp and the controls (tRNA^Leu and tRNA^Tyr), respectively. For each lane, the sum of aminoacylated and deacylated tRNA was set to 100%. (D) Same as C, but analysis was done for the Tb-TrpRS2 RNAi cell line.

Fig. 4.

Ablation of Tb-TrpRS2 selectively abolishes OXPHOS. Succinate and α-ketoglutarate mitochondrial ATP production in crude mitochondrial fractions of the Tb-TrpRS2 RNAi cell line. Uninduced cells (−Tet) are shown on the left, and induced cells (+Tet) are shown on the right of the graphs. The substrate tested is indicated at the top, and additions of antimycin (antim.) and atractyloside (atract.) are indicated at the bottom. ATP productions in mitochondria isolated from uninduced cells tested without antimycin or atractyloside are set to 100%. The bars represent means expressed as percentages from three or more independent inductions. SEs are indicated.

Fig. 5.

Tb-TrpRS1 and Tb-TrpRS2 have distinct substrate specificities. (A) Mitochondrial editing and modification events of the anticodon loop of the tRNA^Trp as described for Leishmania (14). The percentages of the thiolated U (s²U) and the C→U editing as determined for T. brucei tRNA^Trp are indicated. (B Left) The percentage of thiomodified mitochondrial tRNA^Trp of T. brucei was measured by N-acryloylaminophenylmercuric chloride gel electrophoresis (33) and Northern blot hybridization. The shifted band represents thiolated tRNA^Trp(S²U). (B Right) The fraction of thiolated mitochondrial tRNA^Trp that remains thiolated after H₂O₂ treatment was determined as in the left lane. (C) Quantitative RT-PCR assay for edited tRNA^Trp. (C Upper) The cytosolic and mitochondrial tRNA fractions that were used as templates are free of DNA. (C Lower) The blot shows that RNA editing can be analyzed by a restriction digest because it destroys a HinfI site that is present in the cDNA derived from the unedited tRNA^Trp (14). Introduction of a synthetic HinfI plus 20 flanking nucleotides at the 5′ end of the 5′ RT-PCR primer provides an internal control for the HinfI digestion, allowing the quantitative determination of RNA editing. cDNA amplified from unedited tRNA^Trp contains two HinfI sites and, thus, will be digested into three fragments (46, 22, and 21 nt; unedited). The cDNA derived from edited tRNA^Trp contains the synthetic HinfI site only and will be digested into two fragments (68 and 21 nt; edited). Measuring the intensities of the diagnostic bands for nonedited (46 nt) and edited (68 nt) tRNA^Trp allows, after correcting for their different molecular mass, determination of the fraction of edited tRNA^Trp in T. brucei mitochondria. (D) In vitro aminoacylation assays by using [³H]tryptophan and recombinant Tb-TrpRS1 as well as Tb-TrpRS2 as enzymes. (Left and Center Left) Untreated and H₂O₂-treated cytosolic tRNA were charged. (Right and Center Right) Same as Left and Center Left but with untreated and H₂O₂-treated mitochondrial tRNAs. For each graph, the tRNA charged by the Tb-TrpRS2 was set to 100%. The percentage of mitochondrial tRNA^Trp that is thiolated in the untreated and treated fractions is indicated on the right. The means and SE for three independent experiments are shown for aminoacylation of untreated tRNAs. For experiments using H₂O₂-treated tRNAs, the mean of two experiments is shown. The two values each for cytosolic and mitochondrial tRNAs varied by 20% and 10%, respectively. (E) Aminoacylation of T. brucei tRNA^Trp overexpressed in E. coli by using 200 nM enzyme and 0.25 μg/μl tRNA. (Right) Tb-TrpRS1 charging of total E. coli tRNA with T. brucei tRNA^Trp_UCA overexpressed (■), total E. coli tRNA with T. brucei tRNA^Trp_CCA overexpressed (♦), and total E. coli tRNA (▴). (Left) Tb-TrpRS2 charging of total E. coli tRNA with T. brucei tRNA^Trp_UCA overexpressed (■), total E. coli tRNA with T. brucei tRNA^Trp_CCA overexpressed (♦), and total E. coli tRNA (▴).