Journal of Molecular Biology
A Pathogenesis-associated Mutation in Human Mitochondrial tRNALeu(UUR) Leads to Reduced 3′-End Processing and CCA Addition
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.).
References (54)
- et al.
Towards understanding human mitochondrial leucine aminoacylation identity
J. Mol. Biol.
(2003) - et al.
Impairment of tRNA processing by point mutations in mitochondrial tRNALeu(UUR) associated with mitochondrial diseases
FEBS Letters
(1998) - et al.
Modification defect at anticodon wobble nucleotide of mitochondrial tRNAsLeu(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes
J. Biol. Chem.
(2000) - et al.
Abnormal RNA processing associated with a novel tRNA mutation in mitochondrial DNA. A potential disease mechanism
J. Biol. Chem.
(1993) - et al.
Termination of transcription in human mitochondria: identification and purification of a DNA binding protein factor that promotes termination
Cell
(1989) - et al.
Human mitochondrial tRNA processing
J. Biol. Chem.
(1995) - et al.
A new mtDNA mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)
Biochim. Biophys. Acta
(1991) - et al.
Matrices of paired substitutions show the effects of tRNA D/T loop sequence on Drosophila RNase P and 3′-tRNase processing
J. Biol. Chem.
(1998) - et al.
The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNAHis structure and end-processing
J. Mol. Biol.
(2000) - et al.
Decreased CCA-addition in human mitochondrial trnas bearing a pathogenic A4317G or A10044G mutation
J. Biol. Chem.
(2003)
Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity
FEBS Letters
Sequence and organization of the human mitochondrial genome
Nature
tRNA punctuation model for RNA processing in human mitochondria
Nature
Molecular genetic aspects of human mitochondrial disorders
Annu. Rev. Genet.
Mitochondrial DNA mutations and pathogenesis
J. Bioenerg. Biomembr.
Mitochondrial diseases in man and mouse
Science
Mitochondrial encephalomyopathies:gene mutation
Neuromuscul. Disord.
Human mitochondrial tRNAs in health and disease
Cell. Mol. Life Sci.
Decreased aminoacylation of mutant tRNAs in MELAS but not in MERRF patients
Hum. Mol. Genet.
The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome-associated human mitochondrial tRNALeu(UUR) mutation causes aminoacylation deficiency and concomitant reduced association of mRNA with ribosomes
J. Biol. Chem.
The pathogenic A3243G mutation in human mitochondrial tRNALeu(UUR) decreases the efficiency of aminoacylation
Biochemistry
The final cut. The importance of tRNA 3′-processing
EMBO Rep.
This is the end: processing, editing and repair at the tRNA 3′-terminus
Biol. Chem.
In vitro 3′-end endonucleolytic processing defect in a human mitochondrial tRNASer(UCN) precursor with the U7445C substitution, which causes non-syndromic deafness
Nucl. Acids Res.
Pathology-related substitutions in human mitochondrial tRNAIle reduce precursor 3′ end processing efficiency in vitro
Nucl. Acids Res.
Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR)
Nucl. Acids Res.
Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNALeu(UUR) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes
Mol. Cell. Biol.
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2013, Journal of OtologyIdentification of the novel mutation m.5658T>C in the mitochondrial tRNA(Asn) gene in a patient with myopathy, bilateral ptosis and ophthalmoparesis
2013, Neuromuscular DisordersCitation Excerpt :The change involves the loss of highly conserved A–T base pairing, and in silico simulation predicts a relevant free energy loss (−0.2 kcal/mol) in the acceptor-stem of the tRNA, which could alter the stability of the secondary structure of the molecule as well as the folding of the tRNA into its tertiary structure. It has been reported that mutations occurring in nucleotides situated in the acceptor-stem as well as in other domains of the mt-tRNAs such as the anticodon-loop, T-stem and T-loop affect the 3′ end maturation of the pre-tRNA molecules [5,29]. Interestingly, it was observed for the tRNALeu(UUR) that the closer to the 3′ end cleavage site the mutation was located to, the lower processing efficiency of the tRNAase Z enzyme was found [5].
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2012, Journal of OtologyCitation Excerpt :The primary defect in this mutation was an inefficient aminoacylation of the tRNALeu(UUR) (El Meziane et al., 1998; Chomyn et al., 2000). This mutation also affected the processing of the longer RNA precursors (Levinger et al, 2004; Li and Guan, 2010) and the base post–transcriptional modification of the tRNALeu(UUR) (Yasukawa et al., 2000). In cybrids harboring the nearly homoplasmic 3243A>G mutation, the level of aminoacylated tRNALeu(UUR) was reduced approximately 70% to 75% (Chomyn et al., 2000; Li and Guan, 2010).
A new mitochondrial point mutation in the transfer RNA<sup>Lys</sup> gene associated with progressive external ophthalmoplegia with impaired respiratory regulation
2012, Journal of the Neurological SciencesCitation Excerpt :Hence, as soon as a tRNA is bound by the enzyme, the 3′-end can be used for nucleotide incorporation, resulting in an unaffected kcat. An affinity reduction within the same range as observed for the tRNALys mutant G8299A was also described for other pathogenic mitochondrial tRNA mutations like C3303T, affecting the acceptor stem of tRNALeu [13]. Furthermore, it is highly likely that other tRNA maturation steps and aminoacylation are also affected by this mutation leading to cumulative effects that result in a dramatic reduction of functional tRNALys transcript in mitochondria.