Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import

doi:10.1073/pnas.0804283105

Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import

*Department of Microbiology,
^‡‡Ohio State RNA Group, and
^§§Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210-1292;
^‡Departments of Molecular Biophysics and Biochemistry, and
**Chemistry, Yale University, New Haven, CT 06520; and
^§Institute for Physiological Chemistry, Ludwig-Maximilians-University, Butenandtstrasse 5, 81377 Munich, Germany

Contributed by Dieter Söll, May 2, 2008
↵ ^†M.A.T.R. and J.J.R contributed equally to this work. (received for review March 30, 2008)

Abstract

Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA^Gln into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA_CUG ^Gln and tRNA_UUG ^Gln species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA^Gln import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA^Gln species and a bacterial pre-tRNA^Asp can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.

human
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Footnotes

^††To whom correspondence may be addressed. E-mail: dieter.soll{at}yale.edu or alfonzo.1{at}osu.edu
Author contributions: M.A.T.R., J.J.R., B.K., D.S., and J.D.A. designed research; M.A.T.R., J.J.R., B.K., and S.D.-C. performed research; M.A.T.R., J.J.R., B.K., S.D.-C., A.S.R., D.S., and J.D.A. analyzed data; and M.A.T.R., J.J.R., D.S., and J.D.A. wrote the paper.
↵ ^¶Present address: Institut de Biochimie et Génétique Cellulaires, UMR5095 CNRS/Université Bordeaux 2, 33077 Bordeaux Cedex, France.
↵ ^‖Present address: Cluster of Excellence “Macromolecular Complexes,” Mitochondriale Biologie, Fachbereich Medizin, Johann Wolfgang Goethe–Universität Frankfurt am Main, 60590 Frankfurt am Main, Germany.
The authors declare no conflict of interest.