A common tRNA modification at an unusual location: the discovery of wyosine biosynthesis in mitochondria

Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.


Supplementary Figure 2 | Graphic representation of the T. brucei wybutosinesynthesizing homologs. (a)
The two TYW1 paralogs differ with respect to one protein domain -flavin mononucleotide (FMN) binding domain (or flavodoxin-1) is present at the N terminus of TYW1L and absent in TYW1S. TYW4 and TYW5 are fused in trypanosomes, while in mammals they exist as separate proteins. (b) Subcellular localization prediction by TargetP. mTP: Amino acid score based on known mitochondrial targeting sequences. SP: Strength of any present secretory pathway sequences. Other: Strength of localization prediction toa location other than the mitochondrion or secretory pathway. Loc: Final localization prediction based on the scores of 'mTP', 'SP', and 'Other'. S: secretory pathway. M: mitochondrion. '-' : other. RC (reliability class): Confidence of localization prediction based on the difference between the highest and second highest localization scores. 1 is the highest and 5 is the lowest. TPlen: number of amino acids residues between the N-terminus and a predicted presequence cleavage site. '-' indicates that a cleavage site was not predicted.
Supplementary Figure 3 | Subcellular localization of TbTYW2, TbTYW3A, TbTYW3B, and TbTYW4/5. Western blot analysis of epitope-tagged constructs from total, cytosolic, and mitochondrial protein fractions. Enolase is used as a cytosolic marker and as a means to determine mitochondrial fraction purity. Isd11 serves as both a mitochondrial marker and a control for cytosolic fraction purity. TbTYW2-Myc localizes to both the cytosolic and mitochondrial fractions. TbTYW3A-HA is localized to the cytosol, while TbTYW3B-FLAG is strictly found in the mitochondrion. TbTYW4/5-Myc is strictly cytosolic.

Supplementary Figure 4 | Positive identification of wyosine, and absence of isowyosine, in the trypanosome mitochondrion.
(a) imGpA (wyosine-phosphate adenosine) from Candida utilis elutes at 38.7 minutes (left panel) and has the absorbance profile seen in the right panel. (b) Wyosine from T. brucei elutes at 38.7 minutes and has a matching absorbance profile to the C. utilis wyosine absorbance profile. (c) Combination of C. utilis imGpA fraction and T. brucei suspected imGpA fraction co-elute. The combined C. utilis and T. brucei sample is shifted slightly to the left, likely due to the nearly complete lack of other nucleosides that would influence (retard) the imGpA dinucleoside as it migrates through the reverse-phased HPLC column. (d) and (e) Effects of TbTyW1S and TbTyW1L RNAi on the growth of the procyclic stage of T. brucei in the low glucose media compared to normal media as indicated. Growth curves of wild type (WT; triangles), non-induced (TET-; squares) and induced (TET+; circles) knockdown cell line for TbTyW1S and TbTyW1L. The y axis is labeled by a log scale and represents the products of the measured cell densities and total dilutions. Cell densities were measured using the Beckman Z2 cell counter.

Supplementary Figure 5 | Sequence-determining ions observed in product ion
mass spectra from collision-induced dissociation of L. tarentolae tRNA Phe RNase T1-derived anticodon oligonucleotides. (a) Cytoplasmic tRNA Phe (b) mitochondrial tRNA Phe . The conventional tRNA numbering has been used throughout, yielding the sequence AA(OHyW 37 /yW 37 /imG 37 )AUCUG as indicated. All c-and y-type fragment ions and 4 of 5 possible w-type ions were detected and used to confirm the sequence. Only oligonucleotides covering the anticodon loop including position 37 are shown.

Western Blot
The protein coding regions with different epitope tags, were generated for the other T. brucei wybutosine-synthesizing genes: TYW2-Myc, TYW3A-HA, TYW3B-FLAG, and TYW4-Myc. These constructs were individually transformed into procyclic T. brucei 29-13 cells and clonal cell lines were established as described before. Expression was induced by the addition of tetracycline for 24 hr and either harvested and total cell extracts prepared (as described elsewhere) to use for Western blot analysis.
For each construct, one liter of induced cells were harvested at 1 x 10 7 cells/ml. 50 ml of this culture was sonicated to produce the total cell lysate. The rest of the culture was used to prepare the cytosolic and mitochondrial fractions as described elsewhere (36). Total, cytosolic, and mitochondrial protein fractions (10 mg/lane) were separated on 10% SDS-polyacrylamide gel, blotted, and subjected to Western blot analysis with the following monoclonal mouse primary antibodies: V5 and FLAG (Sigma-Aldrich), Myc (Abcam), HA (Sigma), and His (Santa Cruz). Secondary antimouse IgG antibodies coupled to horseradish peroxidase (GE Healthcare) were use for visualization using the Clarity™ Western ECL system (BioRad) and following the manufacturer's instructions. Rabbit polyclonal antibodies specific for T. brucei Isd11 (28) and enolase (kindly provided by P.A.M. Michels) were used as controls for mitochondrial and cytosolic fraction purity, respectively.