The analysis of tRNA import into mammalian mitochondria.
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.
Mager-Heckel
Anne-Marie
AM
FRE 2375 CNRS MLPH, IPCB, Strasbourg, France.
Entelis
Nina
N
Brandina
Irina
I
Kamenski
Petr
P
Krasheninnikov
Igor A
IA
Martin
Robert P
RP
Tarassov
Ivan
I
D001483:Base Sequence; D015152:Blotting, Northern; D011232:Chemical Precipitation; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D034443:RNA Transport; D000077278:RNA, Mitochondrial; D034741:RNA, Small Interfering / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000077278:RNA, Mitochondrial; D034741:RNA, Small Interfering; D012313:RNA; D012343:RNA, Transfer