Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny

Abstract

Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNAAsp in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNAAsp including predominantly the cloverleaf. On the contrary, the native tRNAAsp folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis–Westhof interactions, the tertiary network core building rules apply to all tRNAAsp from mammalian mitochondria.

A novel mutation in the mitochondrial tRNAAla gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

“…In addition to that, this change could also alter the folding of the tRNA. In bacterial, archaeal and eukaryotic cytosolic tRNAs, the transition from the secondary (cloverleaf) to the tertiary (L-shape) structures is supported by nine interactions involving conserved and semiconserved residues located in the D and T-loops and the core of the molecule (Messmer et al, 2009). The hydrogen bonds between the nucleotides 12 (corresponding to 5644A in mt-tRNA Ala ) and 23 (5636T) participate in this folding; therefore, the loss of these hydrogen bonds might compromise the tertiary structure of the molecule.…”

Genetic aspects of mitochondrial genome evolution

“…Then they are chemically modified to ensure their proper folding, recognition, and basepairing (Helm and Attardi, 2004;Messmer et al, 2009). Modifications of the anticodon stem, such as the hypermodified nucleoside 5-methylaminomethyl-2-thiouridylate at position 34, create the "wobble-base" necessary to enable the recognition of multiple codons (Agris et al, 2007).…”

Translation in Mammalian Mitochondria: Order and Disorder Linked to tRNAs and Aminoacyl-tRNA Synthetases

“…The 3D structure of canonical tRNAs is based on a set of tertiary interactions between nucleotides at distance in the secondary structure, in particular on a network of interactions defining and stabilizing the "central core" of the L-shaped structure. Fine-tuned chemical structural probing on two case studies, human mt-tRNA Asp and bovine mt-tRNA Phe confirmed the existence of these networks (Messmer et al 2009;Wakita et al 1994). It remains to be verified if such networks can take place in all mt-tRNAs, an hypothesis that could not be supported by nucleotide conservation, but that will need a more detailed consideration of nucleotide partnership rules as tackled by Leontis and Westhof (Leontis et al 2002).…”

“…It remains to be verified if such networks can take place in all mt-tRNAs, an hypothesis that could not be supported by nucleotide conservation, but that will need a more detailed consideration of nucleotide partnership rules as tackled by Leontis and Westhof (Leontis et al 2002). Initial data are available suggesting that at least all mammalian mt-tRNA Asp would benefit from the set of tertiary network interactions despite nonconservation of involved nucleotides (Messmer et al 2009). Extension of the analysis to the full set of mammalian mt-tRNAs is in progress.…”

“…Therefore, any insight on the precise rules governing secondary and tertiary folding of the full set of human mt-tRNAs remains of high importance in order to comprehend the high sensitivity of these tRNAs to mutations perturbing their structure. Along these lines, pioneered experiments (Helm et al 1998;Messmer et al 2009), combined with the implementation of dedicated database (Pütz et al 2007), and the development of bioinformatics tools (Bernt et al 2012;Jühling et al 2012a) are opening the path toward a solid knowledge on mt-tRNA 3D structures.…”