The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA

Abstract

Direct sequencing of human mitochondrial tRNALysshows the absence of editing and the occurrence of six modified nucleotides (m1A9, m2G10, Psi27, Psi28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNALys. Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m1A9 in this process.

Alcohol-induced S-adenosylhomocysteine accumulation in the liver sensitizes to TNF hepatotoxicity: Possible involvement of mitochondrial S-adenosylmethionine transport

“…In addition to the synthesis of phospholipids, SAM is essential for the methylation of various types of nucleic acids present in the mitochondria. In mammals, methylation of DNA, tRNA and rRNA has been documented, and some have been shown to be essential for mitochondrial function [44][45][46][47]. SAM is also necessary for post-translational modifications of some mitochondrial proteins.…”

Aminoacylation properties of pathology-related human mitochondrial tRNALysvariants

“…Previous work showed that an in vitro transcript of this tRNA does not fold into a functional cloverleaf but into alternative extended bulged hairpins ( Fig. 1A; Helm et al 1998), and is poorly aminoacylated in the presence of a human mt crude enzymatic extract or of an overexpressed and purified mt LysRS (Tolkunova et al 2000). The requirement of a single post-transcriptional modification for cloverleaf folding of tRNA Lys was confirmed by synthesis and structural analysis of a chimeric tRNA Lys , which displays a unique modified base, a methylated adenosine at position 9 ( Fig.…”

“…Such variants, carrying an altered base pair at position 50-64 replacing A50-U64 by U50-A64 (Ki) or by G50-C64 (Ke), adopt the expected cloverleaf folding, as demonstrated by enzymatic and chemical probing ( Fig. 1C; Helm et al 1998).…”

“…tRNA domains are labeled for Ke. Structural mutations introduced at positions 50 and 64 (indicated by gray circles) prevent alternate folding into extended hairpin (black crosses; Helm et al 1998). levels could be established. Figure 3 shows that both native tRNA Lys s are lysylated to about the same level (57 ± 2%).…”

“…This modification impairs the formation of a Watson-Crick base pair between nucleotides 9 and 64 responsible for the extension of the amino acid acceptor stem within the transcript. Interestingly, the structural chaperone effect of m 1 A9 modification can be simulated by mutating either nucleotide 9 or nucleotide 64 (provided base-pairing between residues 64-50 is conserved; Helm et al 1998). Such variants, carrying an altered base pair at position 50-64 replacing A50-U64 by U50-A64 (Ki) or by G50-C64 (Ke), adopt the expected cloverleaf folding, as demonstrated by enzymatic and chemical probing ( Fig.…”