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 modified1. 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 pathways2. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro3. 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 enzyme4, 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’5 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.

In all organisms tRNAs play the essential role of connecting the genetic information found in DNA with the protein synthesis machinery ensuring fidelity during translation. Following transcription tRNAs undergo a number of processing events including numerous post-transcriptional modifications that render a tRNA molecule fully functional. The effects of some modifications go beyond simply affecting tRNA structure and can alter the meaning of the tRNA. This review will summarize the current state of the tRNA editing field, highlighting how editing affects tRNA structure and function in various organisms. It will also discuss recent data that hints at connections between editing and modification that may be exploited by cells to modulate a tRNA’s role in translation.

All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions in translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m 1 G 37 ) of the anticodon loop in several tRNAs, is evolutionarily conserved and participates in translational reading frame maintenance. In eukaryotes, the tRNA methyltransferase TRM5 is responsible for m 1 G formation in nucleus-encoded as well as mitochondria-encoded tRNAs, reflecting the universal importance of this modification for protein synthesis. However, it is not clear what role, if any, mitochondrial TRM5 serves in organisms that do not encode tRNAs in their mitochondrial genomes. These organisms may easily satisfy the m 1 G 37 requirement through their robust mitochondrial tRNA import mechanisms. We have explored this possibility in the parasitic protist Trypanosoma brucei and show that down-regulation of TRM5 by RNAi leads to the expected disappearance of m 1 G 37 , but with surprisingly little effect on cytoplasmic translation. On the contrary, lack of TRM5 causes a marked growth phenotype and a significant decrease in mitochondrial functions, including protein synthesis. These results suggest mitochondrial TRM5 may be needed to mature unmethylated tRNAs that reach the mitochondria and that could pose a problem for translational fidelity. This study also reveals an unexpected lack of import specificity between some fully matured and potentially defective tRNA species.

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.

SUMMARY In cells, tRNAs are synthesized as precursor molecules bearing extra sequences at their 5′ and 3′ ends. Some tRNAs also contain introns which, in archaea and eukaryotes, are cleaved by an evolutionarily conserved endonuclease complex that generates fully functional mature tRNAs. In addition, tRNAs undergo numerous post-transcriptional nucleotide chemical modifications. In Trypanosoma brucei the single intron-containing tRNA (tRNATyrGUA) is responsible for decoding all tyrosine codons; therefore, intron removal is essential for viability. We show by molecular and biochemical approaches the presence of several non-canonical editing events, within the intron of pre-tRNATyrGUA, involving guanosine to adenosine transitions (G to A) and an adenosine to uridine transversion (A to U). The RNA editing described here is required for proper processing of the intron, establishing for the first time the functional significance of non-canonical editing with implications for tRNA processing in the deeply divergent kinetoplastid lineage and eukaryotes in general.