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Pathology-Related Mutation A7526G (A9G) Helps in the Understanding of the 3D Structural Core of Human Mitochondrial Trnaasp

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Pathology-Related Mutation A7526G (A9G) Helps in the Understanding of the 3D Structural Core of Human Mitochondrial Trnaasp Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press LETTER TO THE EDITOR Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNAAsp MARIE MESSMER, AGNE`S GAUDRY, MARIE SISSLER, and CATHERINE FLORENTZ Architecture et Re´activite´ de l’ARN, Institut de Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique, Universite´ de Strasbourg, 67084 Strasbourg, France ABSTRACT More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNAAsp to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNAAsp with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency. Keywords: mammalian mitochondria; tRNA; structure probing in solution; pathology-related mutations; mitochondrial disorder INTRODUCTION (e.g., Helm et al. 2000), pathologic or polymorphic inci- dence of a mutation can still not be predicted (Florentz and The human mitochondrial (mt) genome codes for 13 pro- Sissler 2001; Florentz et al. 2003; Levinger et al. 2004; teins, 2 rRNAs, and 22 tRNAs. The 13 proteins are subunits McFarland et al. 2004). Knowledge on three-dimensional of the respiratory chain complexes and contribute to ATP (3D) folding rules is scarce. synthesis. The 22 tRNA genes are hotspots for mutations, Human mt-tRNAAsp is a typical representative of the which are either polymorphisms (neutral consequences) or structural degeneration of mammalian mt-tRNAs. Like 14 related to diseases (see the MITOMAP database) (Ingman out of the 22 mt-tRNAs, it has a biased A-, U-, and C-rich and Gyllensten 2006). Understanding the molecular impact nucleotide content. It can fold into a theoretical secondary of mutations on the multiple facets of tRNA biogenesis, cloverleaf structure but with small D- and T-loops, and structure, and function has been tackled by several com- several conserved nucleotides that would be expected to plementary in vitro and in vivo studies. The mosaic of induce and stabilize the 3D fold (Helm et al. 2000) char- results collected so far favors diverse molecular impacts, acteristic of classical tRNAs (Juehling et al. 2009) appear to with, in some cases, cumulative effects (Wallace et al. 1999; be absent. The gene for this tRNA is affected by mutation Florentz et al. 2003; Jacobs 2003; DiMauro 2006; Shapira A7526G in a patient with a mitochondrial myopathy 2006). Due to the lack of knowledge on structure/function (Seneca et al. 2005). The molecular impact of this mutation relationships of mammalian mt-tRNAs, already recognized is unknown but raises an interesting structural question. as ‘‘bizarre’’ at the primary and secondary structural levels Indeed, mutation A7526G generates a nucleotide transi- tion at a position of potential structural importance for the folding and the 3D structure of the RNA, namely position 9 Reprint requests to: Catherine Florentz, Architecture et Re´activite´ de l’ARN, Institut de Biologie Mole´culaire et Cellulaire, Centre National de la (classical tRNA numbering, Juehling et al. 2009). Investi- Recherche Scientifique, Universite´ de Strasbourg, 15 Rue Rene´ Descartes, gation of this mutation should assist not only in gaining a 67084 Strasbourg, France; e-mail: [email protected]; fax: 33-3- better understanding of the initial molecular impact respon- 88-60-22-18. Article published online ahead of print. Article and publication date are sible for pathology but also in gaining a better knowledge of at http://www.rnajournal.org/cgi/doi/10.1261/rna.1626109. rules governing human mt-tRNA 3D structures. 1462 RNA (2009), 15:1462–1468. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2009 RNA Society. Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Mutation A7526G affects mt-tRNA structure Here, we present a mutagenic analysis involving func- present in the wild-type triple A9/A12-U23. To investigate tional and structural studies in solution on human mt- this hypothesis, the structure as well as the function of tRNAAsp derivatives. Enzymatic structural probing on both wild-type and mutant G9/A12-U23 mt-tRNAAsp were com- wild-type and mutant in vitro transcripts reveals a primary pared. Solution structures were investigated by enzymatic impact of mutation A7526G on structure, which subse- probing while the aminoacylation properties were estab- quently hampers aminoacylation properties of mt-tRNAAsp. lished in the presence of recombinant human mt aspartyl- Compensatory mutations restore both activity and struc- tRNA synthetase (mt-AspRS). Two additional variants, ture and strongly support the existence of a tertiary inter- tRNAAspG9/G12-C23 (with a double transition in the action between nucleotide 9 and base-pair 12-23 within the D-stem) and variant tRNAAspG9/U12-A23 (with a dou- D-stem. Compilation of sequences within the specifically ble transversion) were also studied. dedicated database ‘‘Mamit-tRNA’’ (Pu¨tz et al. 2007) ex- tends the existence of this tertiary interaction to 97.8% Comparative structural investigation of all known mammalian mt-tRNAAsp sequences and to a large group of additional mammalian mt-tRNAs. Structural probing of the transcripts was performed with nuclease S1 (cleaves single-stranded regions) and ribonu- clease V1 (cleaves double-stranded or highly structured re- RESULTS AND DISCUSSION gions) (Giege´ et al. 1999) on 59-end labeled tRNAs. Typical cleavage profiles are shown in Figure 2. They fit with Predicted impact of mutation A7526G (A9G) cloverleaf structures of the RNAs, stems being cleaved by on mt-tRNAAsp structure ribonuclease V1 and loops by nuclease S1. Interestingly, In classical tRNAs (i.e., from bacteria, archae, and cytosol only subtle but significant differences in cleavage intensities of eukaryotes), nine tertiary interactions sustain the 3D appear. There is no general structural breakdown for the fold (Fig. 1), three in and between D- and T-loops, and six pathology-related mutant. Differences in profiles are high- forming the core of the structure (Kim et al. 1974; Westhof lighted in Figure 3, A–D. None of the phosphodiester bonds et al. 1985; for review, see Giege´ et al. 1993; Dirheimer et al. located 39 of residues 9, 12, and 23, are cleaved by nuclease 1995). Myopathy-related mutation A7526G (Seneca et al. S1, but all are cleaved by ribonuclease V1. This is in agree- 2005) affects nucleotide at position 9 in tRNAAsp, poten- ment with involvement of the three nucleotides in a higher- tially part of the 3D network. Mutation A9G (mutant G9/ order structure, in wild type as well as in each of the three A12-U23) would break the link between residues 9 and 23 other molecules. Focus on base-pair 12-23 in the D-stem reveals stronger sensitivity to V1 ribonuclease in mutant mt-tRNAAspG9/A12-U23 and in variant mt-tRNAAspG9/ G12-C23, as compared with the cleavage pattern in wild- type tRNAAsp and variant G9/U12-A23. This is in favor of a local structural effect of mutation A9G on base-pair 12-23. Furthermore, intensity of cleavages is affected at a distance from the mutation as well in the acceptor stem, the T-domain, and the anticodon arm as within the D-domain. The four tRNAs investigated sort into two sets based on their nuclease susceptibilities, wild-type and variant mt-tRNAAspG9/G12-C23 on one hand, and mutant mt- tRNAAspG9/A12-U23 and variant mt-tRNAAspG9/U12-A23 on the other. The first tRNAs (Fig. 3A,C) show strong V1-cuts within the acceptor stem (residue 68), anticodon arm (residues 28, 29, and 30), and residue 25 (next to con- nector 2), as well as weak V1-cuts within the T-loop FIGURE 1. Tertiary interaction network in canonical tRNAs. (A) (residue 56). The second tRNAs (Fig. 3B,D) exhibit the Secondary cloverleaf structure. (B) Tertiary ‘‘L-shaped’’ structure. opposite strength in cleavage patterns, namely, weak Structural domains are indicated, and the tertiary interaction network V1-cuts within the acceptor stem (residue 68), anticodon is emphasized by dotted lines and boldface characters (for review, see arm (residues 28, 29, and 30), and residue 25 and a strong Giege´ et al. 1993; Dirheimer et al. 1995). Nucleotide positions involved in the nine tertiary interactions are either strictly conserved, V1-cut within the T-loop (residue 56). The opposite ribo- semiconserved (Y for pyrimidine, R for purine), or not conserved nuclease V1 reactivity within the two sets is emphasized (black dots). Other strictly conserved or semiconserved nucleotides on 3D folds (Fig. 3E,F) and suggests differential overall are indicated by gray letters. Numbering is according to Juehling et al. flexibilities. Perturbations within the 9/12-23 interaction (2009). Residues 8, 9, and 26 form connectors between domains. Residue 9 forms a triple by interaction with residue 23 of base-pair influence stacking interactions downstream of the anti- 12-23. codon arm, upstream of base pair 12-23, and within the www.rnajournal.org 1463 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Messmer et al. values in the same range, i.e., KM of 1–2 mM and kcat of 100 3 10À3 s-1).
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