Decreased CCA-addition in Human Mitochondrial tRNAs Bearing a Pathogenic A4317G or A10044G Mutation

Yukihide Tomari,Narumi Hino,Takashi Nagaike,Tsutomu Suzuki,Takuya Ueda

doi:10.1074/jbc.m213216200

Yukihide Tomari, Narumi Hino + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m213216200

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Abstract

Pathogenic point mutations in mitochondrial tRNA genes are known to cause a variety of human mitochondrial diseases. Reports have associated an A4317G mutation in the mitochondrial tRNA(Ile) gene with fatal infantile cardiomyopathy and an A10044G mutation in the mitochondrial tRNA(Gly) gene with sudden infant death syndrome. Here we demonstrate that both mutations inhibit in vitro CCA-addition to the respective tRNA by the human mitochondrial CCA-adding enzyme. Structures of these two mutant tRNAs were examined by nuclease probing. In the case of the A4317G tRNA(Ile) mutant, structural rearrangement of the T-arm region, conferring an aberrantly stable T-arm structure and an increased T(m) value, was clearly observed. In the case of the A10044G tRNA(Gly) mutant, high nuclease sensitivity in both the T- and D-loops suggested a weakened interaction between the loops. These are the first reported instances of inefficient CCA-addition being one of the apparent molecular pathogeneses caused by pathogenic point mutations in human mitochondrial tRNA genes.

Highlights

Mitochondrial1 DNA mutations are known to be associated with a variety of human diseases
Effects of Pathogenic A4317G and A10044G Mutations in mt tRNAs on CCA-addition—We found previously [8] that the human mt CCA-adding enzyme requires the T-arm region of mt tRNAs for efficient CCA-addition, prompting us to hypothesize that some mitochondrial diseases associated with pathogenic point mutations in tRNA genes may result from an absence or an inefficiency of CCA-addition to mt tRNA during maturation
Because two pathogenic point mutations, A4317G and A10044G, were found at similar positions in the respective T-loop of mt tRNAIle and mt tRNAGly, we focused our attention on evaluating these mutations (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—[␣-32P]ATP (110 TBq/mmol) was obtained from Amersham Biosciences and [5Ј-32P]pCp (111 TBq/mmol) from PerkinElmer Life Sciences. The 10-␮l reaction mixtures contained 50 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 100 mM KCl, 0.1 mM CTP, and/or ATP, 0.033 ␮M [␣-32P]CTP or [␣-32P]ATP, 1 ␮M substrate tRNA, and 10 ng of purified recombinant CCA-adding enzyme. Determination of Kinetic Parameters for C-addition or A-addition— The 10-␮l reaction mixtures for C-addition or A-addition contained 50 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 100 mM KCl, 10 ng of CCA-adding enzyme, 0.5 mM CTP or ATP, 0.033 ␮M [␣-32P]CTP or [␣-32P]ATP, and 1–12 ␮M tRNA-DC or tRNA-DCC. Conformational Analysis of Small RNA Fragments—Short RNA fragments containing the T-arm region of the wild type and A4317G tRNAIle were transcribed in vitro [14] using the following DNA fragments as templates, and were purified with super-denaturing PAGE (15% polyacrylamide, 7 M urea, 30% formamide, 1ϫ TBE). Measurement of RNA Melting Profiles—Melting profiles were measured by a Gilford Response II spectrophotometer using 0.1 or 0.2 A260 units of RNA samples in 400 ␮l of a buffer consisting of 50 mM sodium cacodylate (pH 7.0), 10 mM MgCl2, and 200 mM NaCl as described previously [16]

RESULTS

Km kcat

DISCUSSION