Aminoacylation properties of pathology-related human mitochondrial tRNA^Lysvariants

Abstract

In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA Lys deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m 1 A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA Lys gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA Lys variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C•A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000-to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.

Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture

“…Whereas variant U54C behaves similarly to wildtype mt-tRNA Tyr , substitutions of G15 by A, and especially of A22 by G, significantly affect the tyrosylation capacity of both molecules with losses in catalytic efficiency (k cat /K M ) of 40-and 600-fold, respectively. Kinetic parameters revealed a predominant effect on k cat (K M only being affected up to sixfold) in accordance with that observed on other aminoacylation systems (e.g., Kelley et al 2000;Sissler et al 2004). Since direct interaction between residues 15, 22, and 54 and the cognate dimeric mt-TyrRS is unlikely (Fig.…”

“…2B). This interpretation is in line with the ranking of this molecule among the ''heavy'' mt-tRNAs with an excess of G-residues (on average 27%) as opposed to the ''light'' species (on average 14%) , which consequently fold into less stable structures as, for example, human tRNA Lys (Sissler et al 2004). …”

Extensive and rapid screening for major mitochondrial DNA point mutations in patients with hereditary hearing loss

“…The m.8348A4G homoplasmic transition is one of the characteristic mtSNPs of mitochondrial haplogroup H1b, and the pathogenicity of this mutation still remains controversial. 41,42 The clinical course of this patient must therefore be followed closely and further detailed investigation is required.…”

Pathogenic mechanism of a human mitochondrial tRNA Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

“…Diseases associated with hmt-tRNA point mutations include MERRF (myoclonic epilepsy with ragged red fibers), MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), and deafness (2)(3)(4). Pathogenic mutations of hmt-tRNAs have been shown to affect global tRNA structure (5)(6)(7)(8), tRNA processing (9-13), modification (14)(15)(16)(17)(18)(19)(20), aminoacylation (7,(21)(22)(23)(24)(25)(26), and translation efficiency (27)(28)(29). However, little is known about the precise pathogenic mechanisms of the vast majority of hmt-tRNA mutations, hindering the development of appropriate treatments.…”