Thiolation Controls Cytoplasmic tRNA Stability and Acts as a Negative Determinant for tRNA Editing in Mitochondria

Abstract

Kinetoplastids encode a single nuclear tryptophanyl tRNA that contains a CCA anticodon able to decode the UGG codons used in cytoplasmic protein synthesis but cannot decode the mitochondrial UGA codons. Following mitochondrial import, this problem is circumvented in Trypanosoma brucei by specifically editing the tRNA Trp anticodon to UCA, which can now decode the predominant mitochondrial UGA tryptophan codons. This tRNA also undergoes an unusual thiolation at position 33 of the anticodon loop, the only known modification at U33 in any tRNA. In other organisms, tRNA thiolation is mediated by the cysteine desulfurase, Nfs1 (IscS). However, T. brucei encodes two Nfs homologues, one cytoplasmic and the other mitochondrial. We show by a combination of RNA interference and Northern and Western analyses that the mitochondria-targeted TbNfs, and not TbNfs-like protein, is essential for thiolation of both cytosolic and mitochondrial tRNAs. Given the exclusive mitochondrial localization of TbNfs, how it mediates thiolation in the cytoplasm remains unclear. Furthermore, thiolation specifically affects thiolated tRNA stability in the cytoplasm but more surprisingly acts as a negative determinant for the essential C to U editing in T. brucei. This provides a first line of evidence for mitochondrial C to U editing regulation in this system.

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

“…Tight regulation of tRNA editing might be required if unedited and edited tRNA Trp have non-overlapping functions in translation assigned to UGG and UGA codons, respectively. We have shown that ablation of Mtu1 essentially abolishes thiolation of mitochondrial tRNA Trp , which in agreement with the results by Wohlgamuth-Benedum et al (31) leads to an increase in C to U editing of the wobble nucleotide. However, ablation of Mtu1 did not effect growth of insect stage T. brucei even though the energy metabolism of these cells depends on oxidative phosphorylation and therefore requires mitochondrial gene products.…”

“…It has been suggested that in T. brucei cytosol-specific thio modifications influence the stability of tRNA Glu and tRNA Gln (31). We could confirm this observation in our RNAi cell lines, however, the down-regulation was more variable and less pronounced.…”

“…Thus, it appears that thiolation at position 33 has an inhibitory effect on the C to U editing of the adjacent uridine. These results are in complete agreement with the recently published study by WohlgamuthBenedum et al (31), which demonstrated the same inhibitory effect of thiolation on editing of trypanosomal tRNA…”

“…We could confirm this observation in our RNAi cell lines, however, the down-regulation was more variable and less pronounced. Wohlgamuth-Benedum et al (31) furthermore showed that down-regulation of thiolation by ablation of Nfs1 causes stimulation of tRNA editing. Based on this result they proposed that the mitochondria-specific thiolation of the tRNA Trp plays a role in regulating the extent of editing.…”

The Fe/S Cluster Assembly Protein Isd11 Is Essential for tRNA Thiolation in Trypanosoma brucei

“…We have recently shown that in the insect stage of the parasite Nfs is indispensable for thiolation of both cytosolic and mitochondrial tRNAs. Furthermore, thiolation has important implications for cytoplasmic tRNA stability of normally thiolated tRNAs and mitochondrial C to U editing of tRNA Trp (24).…”