Mitochondrial tRNA import in Trypanosoma brucei is independent of thiolation and the Rieske protein

Abstract

Due to a complete lack of the tRNA genes in the mitochondrial genome of Trypanosoma brucei, all tRNAs needed for mitochondrial translation have to be imported into the organelle from the cytosol. A previous study showed that the modified nucleotide s 2 U could act as a negative determinant for mitochondrial tRNA import in another kinetoplastid, Leishmania tarentolae. We have investigated whether the same type of cytosolic control for tRNA retention exists in T. brucei. Based on Northern analysis with subcellular RNA fractions and in vitro import assays, we demonstrate that silencing of the cysteine desulfurase, TbNfs (TbIscS), the key enzyme in tRNA thiolation (s 2 U) and Fe-S cluster formation in vivo, has no effect on tRNA partitioning. This observation is especially surprising in light of a recent report suggesting that in L. tropica the Rieske Fe-S protein is an essential component of the RNA import complex (RIC). In line with the above observation, we also show that down-regulation of the Rieske protein by RNA interference, similar to the TbNfs knockdowns, has no effect on import. The data presented here supports the view that in T. brucei: (1) s 2 U is not a negative determinant for tRNA import; (2) the Rieske protein is not an essential component of the import machinery, and (3) since the Rieske protein is essential for respiration and maintenance of inner mitochondrial membrane potential, neither process plays a critical role in tRNA import. We therefore suggest that the T. brucei import machinery differs substantially from what has been described in Leishmania.

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

“…They were able to show that ablation of Tb-Nfs1 by RNAi does not change the extent of mitochondrial localization of thio-modified or non-thio-modified trypanosomal tRNAs. Further experiments in the same study showed that a chemically dethiolated tRNA Glu was as efficiently imported into isolated mitochondria as its fully modified counterpart (34). These results are fully consistent with our results and support the conclusion that in T. brucei thio modifications do not act as antideterminants for mitochondrial tRNA import.…”

“…The same conclusion was reached in a recent publication by Paris et al (34). They were able to show that ablation of Tb-Nfs1 by RNAi does not change the extent of mitochondrial localization of thio-modified or non-thio-modified trypanosomal tRNAs.…”

“…Different import efficiencies for the two tRNA Glu species can be excluded based on the in vitro import assays shown in Fig. 5B as well as based on data shown by Paris et al (34). Moreover, should selective intramitochondrial degradation of the thiolated tagged tRNA Glu be responsible for the observed accumulation of non-thiolated tRNA in mitochondria, we would expect the percentage of the tagged mitochondrial tRNA Glu to decrease with time, because with time an increasingly larger fraction of the molecules would be degraded.…”

The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function

“…Consequently, some, if not all, imported tRNAs have two sets of modifications. A typical tRNA destined for the mitochondrion receives some modifications in the nucleus after transcription, additional modifications in the cytosol, and then more modifications following import into the mitochondrion (Paris et al 2009;Phizicky and Alfonzo 2010). This then implies that in common with organisms that encode most or all of their mitochondrial tRNAs in the organellar genome, T. brucei still imports a subset of nucleus-encoded modification enzymes.…”

“…A similar observation was made in T. brucei using enzymatic sequencing of tRNA Lys , tRNA Leu , and tRNA Tyr , each of which bears a mitochondrion-specific ribose methylated cytidine residue at position 32 (C m 32) of the anticodon (Schneider et al 1994a,b). Likewise, thiolation of tRNAs has been implicated as a negative determinant for tRNA import in the parasitic flagellate Leishmania tarentolae (Kaneko et al 2003), but the same is not true in T. brucei (Paris et al 2009). …”