The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria

Abstract

tRNAs universally carry a CCA nucleotide triplet at their 3′-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

tRNA 3′ shortening by LCCR4 as a response to stress in Trypanosoma brucei

“…To investigate if the CAE is recruited to SGs during nutritional stress and therefore unable to repair the shortened tRNAs, cells expressing a GFP-tagged inducible copy of the CAE were subjected to stress and the localization of the CAE analyzed by immunofluorescence. Supplementary Figure S1D shows that nutritional stress does not trigger the accumulation of the CAE in SGs and that the enzyme remains ubiquitously distributed as previously described ( 20 ). Altogether these data demonstrate that the amount as well as the enzymatic activity and localization of the CAE are not affected during nutritional stress.…”

“…In trypanosomes tRNA genes do not encode the CCA-tail and therefore this sequence has to be added posttranscriptionally by the tRNA nucleotidyltransferase, also referred to as CCA-adding enzyme (CAE) ( 20 ). The observed accumulation of tRNAs lacking the CCA-tail could thus be the consequence of a normal CCA-tail turnover but a reduced activity of the CAE during stress.…”

“…When these cells were exposed to nutritional stress lower amounts of shortened tRNAs were observed compared to the control cells expressing normal levels of the endogenous CAE and complete tRNA repair was achieved sooner during stress recovery (Figure 2D ). To provide direct evidence that the CAE is responsible for in vivo tRNA repair a cell line was used that allows inducible depletion of the enzyme by RNAi ( 20 ). Figure 2E shows that cells depleted of the CAE are unable to fully repair the trimmed tRNAs even after 30 min of recovery.…”

“…Generation of the cell lines allowing inducible expression of the tagged tRNA Glu and RNAi of the CCA-adding enzyme have been previously described ( 19 , 20 ).…”

Adaptation of theRomanomermis culicivoraxCCA-adding enzyme to miniaturized armless tRNA substrates

“…Such extremely truncated tRNAs fold into a three-dimensional boomerang-like shape that deviates from the consensus L-form (Jühling et al, 2018). Yet, these organisms encode for a single CCA-adding enzyme that has to act on both cytosolic as well as mitochondrial tRNA pools (Wolfe et al, 1994;Reichert et al, 2001;Nagaike et al, 2001;Tomari et al, 2002;Shikha & Schneider, 2020), and it was shown for the corresponding enzyme of Caenorhabditis elegans that it recognizes mt-tRNAs lacking D-or T-arm (Tomari et al, 2002). Since in R. culicivorax nine mt-tRNAs lack both arms, representing the strongest deviation from the consensus structure (Jühling et al, 2012), we investigated the co-evolution and substrate adaptation of its CCA-adding enzyme.…”