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
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
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DOI：
10.1016/j.bbrc.2011.09.078
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发表时间：
2011-10-22
影响因子：
3.1
通讯作者：
Rustin, Pierre
中科院分区：
文献类型：
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作者：
Loublier, Sandrine;Bayot, Aurelien;Rustin, Pierre
在很大比例的患者中，线粒体呼吸链复合体I遗传缺陷的分子基础仍然未知。在组成这个大复合体的45个亚基中，超过一半的功能未知。了解这些亚基的功能将有助于我们对线粒体生理学的了解，但也可能揭示一些亚基并不是复合物催化活性所必需的。这一发现的一个直接结果是减少了复合体I活性降低的患者需要测序的候选基因的数量。在这项研究中，我们测试了两种不同的方法来稳定地消灭培养细胞中的复合体I亚基。我们首先发现，慢病毒介导的shRNA表达经常导致靶基因之外的其他基因的不可预测的灭绝。这可归因于宿主细胞基因组中不受控制的遗传物质插入。因此，这种方法似乎不适合研究基因的未知功能。接下来，我们发现可以通过在Flp位点（HEK293 Flp- in细胞）直接插入靶向Cl亚基的miR来特异性地灭绝一个Cl亚基基因。通过使用这种策略，我们明确地证明了NDUFB6亚基是复合体I活性所必需的，并定义了适合于在人类细胞中进行系统和稳定地消除不同多余亚基的条件。(C) 2011爱思唯尔公司版权所有。
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 Cl subunit gene by direct insertion of a miR targeting Cl 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. (C) 2011 Elsevier Inc. All rights reserved.