Repair of tRNAs in metazoan mitochondria

Abstract

The integrity of 3'-ends of tRNAs is essential for aminoacylation and consequently for protein synthesis. The CCA-termini are generated and, if truncated by exonucleolytic activity, restored by tRNA nucleotidyltransferase. However, further truncations at the 3'-end can occur by exonuclease activity or during processing of overlapping tRNA primary transcripts in metazoan mitochondria. In the latter case, the upstream tRNA is released in a 3'-truncated form (lacking up to six bases) and subsequently completed. In human mitochondria, tRNA(Tyr)(missing the discriminator nucleotide A(73)) is completed by a discriminator adding activity followed by CCA addition. Since in vivo a high percentage of further 3'-terminally degraded human tRNA(Tyr)transcripts could be observed, it was tested in an in vitro system whether this repair mechanism for tRNA 3'-ends acts also on these further degraded tRNA versions. Additionally, 3'-truncated versions of two non-overlapping mitochondrial tRNAs (tRNA(Thr)and tRNA(Phe)) were examined. The results show that these transcripts can be repaired during incubation. A similar base incorporating activity was observed in mouse mitochondria, indicating that a repair mechanism for the 3'-end of several tRNAs exists in mitochondria of humans and possibly other metazoans which goes beyond the CCA addition.

Figure 1

In vitro base incorporation in 3′-truncated versions of tRNA^Tyr. (A) Incubation of the transcripts with nucleotides in the presence (S) or absence (M) of S100 protein extract. All offered transcripts are accepted as substrates and elongated for several bases, leading to a reduced electrophoretic mobility of the products. (B) Secondary structures of tRNA^Tyr-2 and tRNA^Tyr-4. The asterisk indicates the diagnostic base of the substrate tRNAs, while the human residue at that position is indicated in brackets. The correctly incorporated bases are numbered and drawn in bold characters, the percentage values indicate the fidelity. In the case of tRNA^Tyr-4, only partial addition of the CCA terminus was observed.

Figure 2

Repair of tRNA^Phe-1. (A) ³³P-5′-end-labeled in vitro transcripts representing tRNA^Phe and tRNA^Phe-1 were incubated in the presence of human S100 mitochondrial protein extract and nucleotides, leading to base incorporation and shifted product bands migrating at identical positions (S). In parallel, mock incubation in the absence of protein was performed (M). For determination of the nature of the incorporated nucleotides, the shifted bands were isolated and their 3′-terminal sequence determined (Table 2). (B) Schematic drawing of tRNA^Phe-1 carrying incorporated bases (bold characters). Seventy-eight percent of the analyzed clones showed the correct base A₇₃ added at the discriminator position. Additionally, all sequences carried either the complete or partial CCA terminus. Diagnostic bases (chimpanzee Pan troglodytes sequence) are indicated by asterisks, the nucleotides in the human transcript are shown in brackets.

Figure 3

In vitro base incorporation in tRNA^Thr-1 and tRNA^Thr-2. (A) In the presence of S100 extract and NTPs (S) the tRNA^Thr as well as the truncated versions were extended to the same length, as indicated by identical migration properties. M, mock incubation without S100 extract. (B) Inferred secondary structure of tRNA^Thr-1 showing a low discriminator (A₇₃) repair fidelity of 8%. Diagnostic bases are indicated by asterisks, the original bases in the human tRNA^Thr are shown in brackets.

Figure 4

Dynamic model of tRNA repair. tRNA^Tyr-4 is presented as an example. The 3′-end of the tRNA is subjected to a permanent turnover by elongation and degradation. The incorporation of the correct sequence leading to aminoacylation of the tRNA has two consequences (right). First, the molecule becomes protected against exonucleolytic degradation. Second, translation removes this tRNA from the equilibrium, leading thereby to an increase of repaired tRNA molecules in the pool. In contrast, misincorporation of nucleotides at the truncated 3′-end impedes aminoacylation of the tRNA, which is therefore not protected against exonucleolytic activity (left). After removal of the (incorrect) 3′-terminal nucleotides, the tRNA has a second chance to be elongated correctly.