A Pathogenesis-associated Mutation in Human Mitochondrial tRNALeu(UUR) Leads to Reduced 3′-End Processing and CCA Addition

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Abstract

Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5′-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3′-end processing and CCA addition at the tRNA 3′-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3′-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.

Introduction

The circular DNA located in human mitochondria encodes 13 proteins, two ribosomal RNAs and a set of 22 tRNAs1., 2. which, when complemented with nuclear-encoded enzymes and protein factors, are sufficient for gene expression in these organelles. Since 1988, more than 145 mutations in the mitochondrial genome have been linked with inherited severe neuromuscular diseases†, and more than 90 of them are located in tRNA genes.3., 4., 5., 6., 7. The gene for tRNALeu(UUR) is a hot spot for such pathological mutations, in which 20 disease-correlated base substitutions have been identified. These mutations (especially A3243G, the most studied mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome mutation) cause severe mitochondrial dysfunctions and multisystemic disorders.

One consequence of such mutations can be the reduction in the level of charged tRNA, as observed for A3243G.8., 9., 10., 11. However, tRNA mutations can have effects in addition to reduced aminoacylation. Mitochondrial tRNAs are transcribed as precursors with a 5′-leader, which is removed by RNase P, and a 3′-trailer, which can be endonucleolytically removed by 3′-tRNase.12 Subsequently, the CCA terminus, which is required for aminoacylation, must be added to the 3′-end by tRNA nucleotidyltransferase.13 In vitro studies suggest that each step can be affected by mutations in mitochondrial tRNA genes, leading to reduced expression of the corresponding mature tRNA.14., 15., 16. In addition to the less efficient precursor processing, post-transcriptional base modification can be affected.17., 18. Furthermore, because the mitochondrial genome encodes long polycistronic transcripts punctuated by tRNAs, accurate excision of these tRNAs is also required for rRNA and mRNA maturation.2 Therefore, in addition to defects in tRNA metabolism, tRNA mutations can interfere with precursor excision and consequently affect other aspects of mitochondrial function. Interestingly, three of the tRNALeu(UUR) mutations (A3243G, T3271C, and A3302G) cause an in vivo increase in the steady-state level of a discrete RNA precursor (RNA 19) consisting of the linked 16 S rRNA, tRNALeu(UUR) and ND1 mRNA (illustrated in Figure 1).19., 20., 21.

Furthermore, pathogenic mutations in tRNA genes may not only interfere with the expression or function of the corresponding transcripts, but could also affect the regulation of gene expression in mitochondria. mTERF is a nuclear-encoded protein that specifically binds to mitochondrial DNA within the first 20 bp of the tRNALeu(UUR) gene and triggers termination of H strand transcription at the 3′-end of the large rRNA.22 The pathogenic MELAS mutation A3243G, located in the target DNA sequence for mTERF binding, weakens the mTERF–DNA interaction in vitro,23., 24. suggesting that excessive run-on transcription leading to an excess of large rRNAs with incorrect 3′-ends could cause pathogenesis. However, in vivo analyses showed that the level of both rRNAs encoded upstream of the mTERF binding site was not changed in mitochondria carrying the A3243G mutation, and that mTERF binding is unaffected by the MELAS mutation.9 However, the A3243G mutation could promote the formation of tRNA dimers by Watson–Crick base-pairing of six nucleotides in the D stem, perhaps impeding the correct function of the tRNA.25 Besides these effects, the rate of post-transcriptional base modifications of tRNALeu(UUR) carrying A3243G is reduced and may decrease translational efficiency.17., 26.

Here, the effects of base substitutions in tRNALeu(UUR) on maturation events taking place at the 3′-terminus were more thoroughly investigated: endonucleolytic removal of the 3′-trailer as well as the addition of the invariant CCA terminus were analyzed in several pathogenic tRNALeu(UUR) precursors by in vitro studies using 3′-tRNase from mitoplast extract and a recombinant human mitochondrial tRNA nucleotidyltransferase. The substitutions reduced the efficiency of 3′-tRNase processing below that of the wild-type precursor, and the one with an acceptor stem substitution closest to the processing site also displayed reduced efficiency of CCA addition. These reductions could not be explained by abnormal secondary structures of the transcripts, since structure-probing nucleases did not detect differences in folding between the wild-type and mutant tRNALeu(UUR) precursors, but interestingly, the tRNALeu(UUR) displays an unusual structure through much of the D and anticodon domains.

Section snippets

Mutant tRNALeu(UUR) transcripts as substrates for 3′-tRNase

In the H strand transcript of the human mitochondrial (mt) genome, the gene for tRNALeu(UUR) punctuates the genes for 16 S rRNA and ND1 (Figure 1). Proper mitochondrial function requires precise excision of this tRNA from the precursor transcript, presumably by RNase P cleavage at its 5′-end and by 3′-tRNase at its 3′-end.27 A total of 20 pathogenesis-associated mutations have been found in tRNALeu(UUR), and three of them (A3243G, T3271C and A3302G) increase the steady-state level of the

A series of required tRNA end-processing reactions

Human mitochondrial tRNAs must be excised from long precursor transcripts by RNase P and 3′-tRNase (as illustrated for tRNALeu(UUR) in Figure 1). Following cleavage after the discriminator by 3′-tRNase, tRNA nucleotidyltransferase adds the CCA triplet (which is not encoded in mitochondrial tRNA genes) to the tRNA 3′-ends.13 According to the punctuation model, both the production of mature and functional tRNAs and also of mRNAs and rRNAs require that the excision reactions be accurate and

Conclusions

The experiments presented using 3′-tRNase and tRNA nucleotidyltransferase show only moderately reduced efficiencies of the catalyzed reactions: 3′-tRNase activity decreased up to 3.3-fold, depending on the tRNA mutation, and CCA addition to the C3303T mutant is 5.5-fold reduced compared to wt tRNALeu(UUR). Previous findings are consistent with the hypothesis that endonucleolytic 3′-end processing effects could contribute to mitochondrial pathology: tRNASer(UCN) with the T7445C substitution

Preparation of tRNA precursors

Plasmids carrying tRNALeu(UUR) inserts (wild-type, A3243G, A3302G, and C3303T) linked to a T7 promoter and a hammerhead ribozyme designed to cleave the tRNA at position 111., 53. were adapted for investigating 3′-end processing by replacing the sequence CCA (position 74–76) with the first 38 nt of the 3′-end extension of the human mitochondrial tRNALeu(UUR) precursor followed by a SmaI runoff site (5′-ACATACCCATGGCCAACCTCCTACTCCTCATTGTACCC-3′), and re-cloned into vector pHC624.

Unlabeled RNA was

Acknowledgments

We gratefully acknowledge B. Sohm for donating the initial plasmids and H. Betat, A. Mohan, C. Rammelt, M. Sissler and D. Thurlow for helpful discussions. This work was supported by the Centre National de Recherche Scientifique (C.F. & L.L.), NIH grants S06GM08153 and T34GM08498 (L.L.), the Max Planck Society and by the DFG (grant Mo634/2; I.O. & M.M.), and sabbatical support by NIH fellowship F33-GM64266 and by Université Louis Pasteur, Strasbourg (L.L.).

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