title: The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: a proof of principle study on stable and controlled RNA interference in human cell lines.
abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.
authors.affiliation: Inserm U676, Hôpital Robert Debré, F-75019 Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Loublier
authors.forename: Aurélien
authors.initials: A
authors.lastname: Bayot
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Rak
authors.forename: Riyad
authors.initials: R
authors.lastname: El-Khoury
authors.forename: Paule
authors.initials: P
authors.lastname: Bénit
authors.forename: Pierre
authors.initials: P
authors.lastname: Rustin
mesh_terms: D045744:Cell Line, Tumor; D004579:Electron Transport / Q000235:genetics; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; C527690:NDUFB6 protein, human