Impairment of mitochondrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia

Abstract

We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNA(Ile). The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient's healthy mother. Functional characterization of the mutation revealed a six-fold reduced rate of tRNA(Ile) precursor 3' end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNA(Ile) displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNA(Ile) precursor 3' end processing and contribute to the molecular pathomechanism of this mutation.

Figure 1. Electron microscopy of cross section from limp muscle biopsy

The cross section shows a region of an unaffected and an affected myofiber. The affected fiber shows subsarcolemmal accumulation of mitochondria producing the appearance of ragged red fibers. A significant proportion of mitochondria are enlarged, display with deranged cristae and contain paracrystalline material in the form of the classic “parkin lot” inclusions. The magnified area shows morphologically abnormal mitochondria located between the contractile elements which are absent in the health myofiber.

Figure 2. Analysis of the m.4308G>A mutation in the tRNA^Ile gene

A) Sequence chromatogram of the encompassing the m.4308G>A mutation (arrow) from patient muscle. B) RFLP of the Mutation m.4308G>A in blood and skeletal muscle of the patient and her mother. Corresponding bands to the WT and m.4308G>A substitutions are indicated at right.

Figure 3. tRNase Z^L processing kinetics with the Wild Type and G4308A mutant of human mitochondrially encoded pre-tRNA^Ile

A) Secondary structure of pre-tRNA^Ile. The tRNase Z substrate has a 20 nt 3’ end trailer. The m.4308G>A substitution and the tRNase Z processing site are identified with arrows. B) Processing kinetics of wild type pre-tRNA^Ile and the m.4308G>A mutant with tRNase Z^L. Reactions were performed with unlabeled pre-tRNA^Ile at 2, 5, 10, 20 and 50 nM and with a constant trace concentration of labeled pre-tRNA^Ile. The appropriate concentration of enzyme to use was first determined by analyzing the % product/minute (equivalent to V/[S]) obtained with the wild type and mutant substrates. Reactions were sampled at 5, 10 and 15 minute intervals as indicated by brackets below the panel. -Subst and -Prod designations on the right identify substrate and product bands. C) Eadie-Hofstee plots of wild type tRNA^Ile and the G4308A mutant. Equations are displayed on the Eadie-Hofstee plots (slope is –K_M, y-intercept is V_max, with units of × 10⁻⁸ M and × 10⁻¹⁰ M/min., respectively).

Figure 4. Structure probing of tRNA^Ile wild type and m.4308G>A

Panels A, B: denaturing 6% polyacrylamide gels of pre-tRNA^Ile wild type (left) and the G4308A variant (right). Lanes 0–10 are identified by designations below the gel. Designations at left are nucleotide numbers based on the canonical tRNA postions. Open and filled dots indicate nucleotide positions that display a structure different from the wild type. Panel C: ImageQuant traces of wild type and G4308A cleaved with RNase V1.

Figure 5. The m.4308G>A substitution in tRNA^Ile causes structural rearrangements

A) Changes in V1 susceptibility are identified with dots in the secondary structure. Decreased V1 susceptibility of the G4308A mutant is observed at the site of the substitution and increased V1 susceptibility is observed at U56 in the T loop and U65 at a hinge position between the T arm and acceptor stem. The arrow indicates the 3’ end processing site. Possible structural rearrangements of G4308A tRNA^Ile include B) a long T stem and loop, a reduced variable and practically no acceptor stem and C) a completely open T-arm/Variable arm with an intact acceptor stem. The dashed arrow at the normal tRNase Z cleavage site indicates that tRNase Z would not be expected to cleave a tRNA with either of these structural rearrangements. The three-headed arrow between (A, B, C) indicates a continuum of secondary structures favoring those that would produce an unfavorable substrate for tRNase Z (see text for further interpretation).