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Mitochondria pp 235-253 | Cite as

The Analysis of tRNA Import Into Mammalian Mitochondria

  • Anne-Marie Mager-Heckel
  • Nina Entelis
  • Irina Brandina
  • Petr Kamenski
  • Igor A. Krasheninnikov
  • Robert P. Martin
  • Ivan Tarassov
Part of the Methods in Molecular Biology™ book series (MIMB, volume 372)

Abstract

Ribonucleic acid (RNA) import into mitochondria occurs in a variety of organisms. In mammalian cells, several small RNAs are imported in a natural manner; transfer RNAs (tRNAs) can be imported in an artificial way, following expression of corresponding genes from another organism (yeast) in the nucleus. We describe how to establish and to analyze such import mechanisms in cultured human cells. In detail, we describe (1) the construction of plasmids expressing importable yeast tRNA derivatives in human cells, (2) the procedure of transfection of either immortalized cybrid cell lines or primary patient’s fibroblasts and downregulation of tRNA expression directed by small interfering RNA (siRNA) as a way to demonstrate the effect of import in vivo, (3) the methods of mitochondrial RNA isolation from the transfectants, and (4) approaches for quantification of RNA mitochondrial import.

Key Words

Aminoacylation mitochondrial import real-time quantification siRNA downregulation tRNA 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Anne-Marie Mager-Heckel
    • 1
  • Nina Entelis
    • 1
  • Irina Brandina
    • 1
    • 2
  • Petr Kamenski
    • 1
    • 2
  • Igor A. Krasheninnikov
    • 2
  • Robert P. Martin
    • 1
  • Ivan Tarassov
    • 1
  1. 1.FRE 2375 CNRS “MLPH,” IPCBStrasbourgFrance
  2. 2.Molecular Biology DepartmentBiology Faculty of Moscow State UniversityMoscowRussia

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