Life without post-transcriptional addition of G₋₁: two alternatives for tRNA^His identity in Eukarya

Abstract

The identity of tRNAHis is strongly associated with the presence of an additional 5′-guanosine residue (G−1) in all three domains of life. The critical nature of the G−1 residue is underscored by the fact that two entirely distinct mechanisms for its acquisition are observed, with cotranscriptional incorporation observed in Bacteria, while post-transcriptional addition of G−1 occurs in Eukarya. Here, through our investigation of eukaryotes that lack obvious homologs of the post-transcriptional G−1-addition enzyme Thg1, we identify alternative pathways to tRNAHis identity that controvert these well-established rules. We demonstrate that Trypanosoma brucei, like Acanthamoeba castellanii, lacks the G−1 identity element on tRNAHis and utilizes a noncanonical G−1-independent histidyl-tRNA synthetase (HisRS). Purified HisRS enzymes from A. castellanii and T. brucei exhibit a mechanism of tRNAHis recognition that is distinct from canonical G−1-dependent synthetases. Moreover, noncanonical HisRS enzymes genetically complement the loss of THG1 in Saccharomyces cerevisiae, demonstrating the biological relevance of the G−1-independent aminoacylation activity. In contrast, in Caenorhabditis elegans, which is another Thg1-independent eukaryote, the G−1 residue is maintained, but here its acquisition is noncanonical. In this case, the G−1 is encoded and apparently retained after 5′ end processing, which has so far only been observed in Bacteria and organelles. Collectively, these observations unearth a widespread and previously unappreciated diversity in eukaryotic tRNAHis identity mechanisms.

Identification of distinct biological functions for four 3′-5′ RNA polymerases

“…In D. discoideum , cytosolic and mitochondrial HisRS are encoded by two separate genes. It is possible that D. discoideum mitochondrial HisRS can catalyze sufficient aminoacylation of mt-tRNA His in the absence of G -1 , as already observed for some HisRS enzymes, including the mitochondrial isoform of S. cerevisiae HisRS ( 22 , 38 , 57 ). Interestingly, a precedent for incomplete modification of mt-tRNA His with G -1 has already been established in potato mitochondria, where despite the presence of an encoded G -1 , a substantial fraction of the mt-tRNA His lacks G -1 due to an apparent miscleavage by mitochondrial RNase P ( 58 ).…”

“…In D. discoideum , however, where DdiThg1 is not localized to mitochondria (Figure 1 ), two possible scenarios are suggested. Either mt-tRNA His lacks a G -1 residue, as recently observed for some other protozoan eukaryotes ( 22 , 38 ), or another enzyme such as DdiTLP2 is responsible for the mt-tRNA His G -1 addition reaction.…”

“…Most animals and fungi appear to have lost the ancient TLP enzymes and instead have retained the single eukaryotic Thg1 that was the founding member of the enzyme family. However, D. discoideum is representative of many TLP-containing organisms that are widespread among the eukaryotic family tree, which also includes some species that lack Thg1 entirely and only encode TLP orthologs with as yet undetermined functions ( 10 , 38 ). Therefore, studies in D. discoideum , where both types of enzymes are present and are biologically relevant, promise to provide fertile ground for a deeper understanding of the evolutionary history and biological capabilities of these unusual enzymes.…”

Molecular mechanism of substrate recognition and specificity of tRNAHis guanylyltransferase during nucleotide addition in the 3′–5′ direction

“…In eukaryotes, Thg1 catalyzes the addition of a guanosine residue to the 5 ′ -end of immature tRNA His (Gu et al 2003). This guanosine at position −1 (G −1 ) of tRNA His serves as a major recognition element for histidyl-tRNA synthetase (HisRS) (Cooley et al 1982;Himeno et al 1989;Rudinger et al 1994; Rosen and Musier-Forsyth 2004), except in some rare cases (Rao and Jackman 2015). Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes.…”

MINTmap: fast and exhaustive profiling of nuclear and mitochondrial tRNA fragments from short RNA-seq data

“…In most eukaryotes, the tRNAs encoding the anticodon for histidine (His) require the post-transcriptional addition of a G nucleotide at their 5′ end before they can be recognized by their cognate tRNA synthetase 57 . This post-transcriptionally added nucleotide is described as the “−1 nucleotide” in the literature 58 59 .…”