Structural and mechanistic studies of mammalian mitochondrial RNAs Public Deposited

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  • March 21, 2019
  • Jones, Christie Nicole
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of 9 monocistronic and two dicistronic mRNAs. These mRNAs lack both 5 prime and 3 prime untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5 prime ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than -3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5 prime ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5 prime sequences into the mRNA entrance site to participate in translation initiation. The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNAMet (hmtRNAMet) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNAMet with a C8. Here we show that the single U8C mutation leads to a failure of the tRNA to respond conformationally to Mg2+. This mutation results in a drastic disruption in the structure of the hmtRNAMet, which significantly reduces its aminoacylation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg2+ the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg2+, the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg2+ binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.
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  • In Copyright
  • Spremulli, Linda
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  • University of North Carolina at Chapel Hill
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