The 3' addition of CCA to mitochondrial tRNASer(AGY) is specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1

Abstract

Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.

Figure 1.

Mutational analysis of TRNT1. (A) Schematic representation of the TRNT1 gene showing the position of the mutations identified in this study. (B) Alignment of the amino-acid sequences of TRNT1 homologs in different species showing high degree of evolutionary conservation of the amino-acid residues mutated in the subjects.

Figure 2.

Mitochondrial translation and levels of TRNT1 in the subjects. (A) Pulse-labeling of the mitochondrial translation products in fibroblasts from the subjects and controls, before and after over-expression of wild-type TRNT1. Upper panel. The 13 mitochondrial translation products are indicated at the left of the upper panels: seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX) and two subunits of complex V (ATP). Middle panel: duplicate samples were used for immunoblotting with antibodies against the 70 kDa subunit of complex II to confirm equal loading. Lower panel: the levels of TRNT1 in fibroblasts from the two subjects, and the over-expression of TRNT1 in control and subject fibroblasts were evaluated by immunoblotting with polyclonal antibodies against TRNT1. (B) Quantification of the rate of synthesis of the individual polypeptides in the pulse translation experiments.

Figure 3.

Mitochondrial translation is severely impaired following knockdown of TRNT1 to immunologically undetectable levels. (A) Mitochondrial translation products synthesized in fibroblasts from subjects and controls, before and after transient transfection with a siRNA construct specific to TRNT1. The 13 mitochondrial translation products are indicated at the left of the upper panel as in Figure 1. Middle panel: duplicate samples were used for immunoblotting with antibodies against prohibitin to confirm equal loading. Lower panel: the knockdown of TRNT1 in fibroblasts from the two patients and controls was confirmed by immunoblotting with polyclonal antibodies against TRNT1. (B) Quantification of the rate of synthesis of the individual polypeptides in the pulse translation experiments.

Figure 4.

Differential effect of a destabilizing mutation in TRNT1 on individual tRNAs. Total RNA isolated under acidic conditions from the subject and control fibroblast lines indicated at the top of the figure (12 μg per sample) was separated through a 12% polyacrylamide/8 m urea gel, transferred to a positively charged membrane and hybridized with probes complementary to the mitochondrial and cytoplasmic tRNAs indicated at the right of the figure.

Figure 5.

Endogenous levels of TRNT1 are considerably lower in muscle mitochondria than in fibroblast mitochondria. Immunoblot analysis of TRNT1 in mitochondrial extracts from three control muscle biopsy specimens and three control fibroblast lines. The 70 kDa subunit of complex II and prohibitin were used as loading controls.