Structural probing of a pathogenic tRNA dimer

Abstract

The A3243G mutation within the human mitochondrial (hs mt) tRNALeuUUR gene is associated with maternally inherited deafness and diabetes (MIDD) and other mitochondrial encephalopathies. One of the most pronounced structural effects of this mutation is the disruption of the native structure through stabilization of a high-affinity dimeric complex. We conducted a series of studies that address the structural properties of this tRNA dimer, and we assessed its formation under physiological conditions. Enzymatic probing was used to directly define the dimeric interface for the complex, and a discrete region of the D-stem and loop of hs mt tRNALeuUUR was identified. The dependence of dimerization on magnesium ions and temperature was also tested. The formation of the tRNA dimer is influenced by temperature, with dimerization becoming more efficient at physiological temperature. Complexation of the mutant tRNA is also affected by the amount of magnesium present, and occurs at concentrations present intracellularly. Terbium probing experiments revealed a specific metal ion-binding site localized at the site of the A3243G mutation that is unique to the dimer structure. This metal ion-binding site presents a striking parallel to dimeric complexes of viral RNAs, which use the same hexanucleotide sequence for complexation and feature a similarly positioned metal ion-binding site within the dimeric structure. Taken together, these results indicate that the unique dimeric complex formed by the hs mt tRNALeuUUR A3243G mutant exhibits interesting similarities to biological RNA dimers, and may play a role in the loss of function caused by this mutation in vivo.

FIGURE 1.

Secondary structure of hs mt tRNA^Leu(UUR). (A) Cloverleaf structure of hs mt tRNA^Leu(UUR). The “X” designates the location of the A3243G (A14G) mutation. (B) Schematic representation of the dimeric complex formed by the pathogenic A3243G hs mt tRNA^Leu(UUR) mutant. The mutated base is shown in bold italics.

FIGURE 2.

Enzymatic probing of the wild-type (WT) and mutant A3243G hs mt tRNA^Leu(UUR). (A) Normalized histograms generated from PAGE analysis of enzymatic cleavage of wild-type (dashed) and mutant A3243G (solid) hs mt tRNA^Leu(UUR) using nuclease S1 under nondenaturing conditions. Positions are numbered according to the consensus tRNA numbering system, and bold nucleotides are located within the proposed dimer interface. (B) Histograms generated from PAGE analysis of enzymatic cleavage of wild-type (dashed) and mutant A3243G (solid) hs mt tRNA^Leu(UUR) using RNase T2. (C) 20% denaturing PAGE of nuclease S1 and RNase T2 structural probing experiments. Sample lanes marked A14 represent probing of the wild-type hs mt tRNA^Leu(UUR), while those labeled G14 represent probing results for the mutant A3243G (A14G) hs mt tRNA^Leu(UUR). Lanes marked T1 represent the guanine ladder performed on wild-type hs mt tRNA^Leu(UUR). Lanes marked C are control samples that were not incubated with nucleases. Positions where losses in cleavage efficiency are observed due to dimerization in the mutant structure are identified with arrows. Multiple trials (> 3) of all probing experiments were performed to ensure the reproducibility of the trends shown.

FIGURE 3.

Native gel analysis of the temperature and magnesium dependence on the formation of the A3243G tRNA dimer. (A) Mg²⁺ dependence of tRNA dimer formation. (Inset) 12% PAGE gel run under native conditions with varying [Mg²⁺] added to samples during annealing ([Mg²⁺] = 0, 1, 2, 4, 6, 8, 10, 20, 30, and 40 mM). (B) Time course of dimer formation at 25°C and at 37°C. Multiple trials (> 3) were performed to ensure the reproducibility of the trends shown.

FIGURE 4.

Terbium cleavage results for wild-type and mutant A3243G hs mt tRNA^Leu(UUR). Reactions were performed on both tRNA transcripts under nondenaturing conditions. (A) Gel analysis of terbium probing of wild-type and A3243G hs mt tRNA^Leu(UUR). Probing was performed over a tRNA concentration gradient of 75, 150, 300, and 600 nM tRNA, and samples were analyzed using 20% denaturing PAGE. Lanes representing the control samples for both the wild-type and mutant A3243G tRNAs are labeled A and G, respectively. Lane T1 is a guanine ladder prepared with wild-type tRNA^Leu(UUR), while Alk represents an alkaline ladder. Lanes representing probing results for the wild-type and A3243G samples increase in tRNA concentration from left to right. (B) Bar graphs extracted from PAGE analysis shown in A. Results represent cleavage intensities at each individual position. The solid line to the right of the gel shown in A represents the analyzed area. Results are normalized according to the total signal along the sample lane. Bars represent the results of terbium cleavage for the mutant A3243G (top) and wild-type (bottom) with [tRNA] = 75 nM (black) and 600 nM (gray). Multiple trials (> 3) of all probing experiments were performed to ensure the reproducibility of the trends shown.