Effects on mitochondrial transcription of manipulating mTERF protein levels in cultured human HEK293 cells.
Effects on mitochondrial transcription of manipulating mTERF protein levels in cultured human HEK293 cells.
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DOI：
10.1186/1471-2199-11-72
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发表时间：
2010-09-16
影响因子：
--
通讯作者：
Jacobs HT
中科院分区：
文献类型：
--
作者：
Hyvärinen AK;Kumanto MK;Marjavaara SK;Jacobs HT
根据其在体外的活性，哺乳动物线粒体转录终止因子mTERF被提出通过在线粒体DNA的rDNA片段的下游的高亲和力结合处终止，并选择性地在重链启动子的PH1位点起始来调节线粒体转录。这定义了一个rDNA转录单元，不同于在PH2启动的“全局”重链转录单元。然而，到目前为止，还没有证据表明这两种重链转录单位的相对活性在体内受到mTERF的调节。为了验证这一假设，我们设计了人类hek293来源的细胞，使其过表达或敲低mTERF，并测量了属于不同转录单位的转录本的稳态水平，即PH2转录单位的tRNALeu（UUR）和ND1 mRNA，以及PH1转录单位的tRNALeu + 12S和16S rRNA。在所有测试条件下，16S rRNA和ND1 mRNA的相对水平是相同的，尽管mTERF敲除导致12S rRNA转录本水平增加。tRNAPhe相对于tRNALeu（UUR）的数量不受mTERF过表达的影响，仅因mTERF敲低而略有改变，并且在溴化乙啶诱导的线粒体RNA耗尽恢复期间保持不变。mTERF过表达或敲低会导致其高亲和力结合位点两侧反义转录物相对丰度的显著变化（3-5倍）。mTERF蛋白水平对mtDNA反义链上的读透转录量有实质性影响，而对义链转录的影响是复杂的，并提示了补偿机制的影响。
Based on its activities in vitro, the mammalian mitochondrial transcription termination factor mTERF has been proposed to regulate mitochondrial transcription by favouring termination at its high-affinity binding immediately downstream of the rDNA segment of mitochondrial DNA, and initiation selectively at the PH1 site of the heavy-strand promoter. This defines an rDNA transcription unit distinct from the 'global' heavy-strand transcription unit initiating at PH2. However, evidence that the relative activities of the two heavy-strand transcription units are modulated by mTERF in vivo is thus far lacking. To test this hypothesis, we engineered human HEK293-derived cells for over-expression or knockdown of mTERF, and measured the steady-state levels of transcripts belonging to different transcription units, namely tRNALeu(UUR) and ND1 mRNA for the PH2 transcription unit, and tRNAPhe plus 12S and 16S rRNA for the PH1 transcription unit. The relative levels of 16S rRNA and ND1 mRNA were the same under all conditions tested, although mTERF knockdown resulted in increased levels of transcripts of 12S rRNA. The amount of tRNAPhe relative to tRNALeu(UUR) was unaffected by mTERF over-expression, altered only slightly by mTERF knockdown, and was unchanged during recovery from ethidium bromide-induced depletion of mitochondrial RNA. mTERF overexpression or knockdown produced a substantial shift (3-5-fold) in the relative abundance of antisense transcripts either side of its high-affinity binding site. mTERF protein levels materially affect the amount of readthrough transcription on the antisense strand of mtDNA, whilst the effects on sense-strand transcripts are complex, and suggest the influence of compensatory mechanisms.
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影响因子：
5.6
作者：
GAINES, G;ATTARDI, G
通讯作者：
ATTARDI, G
影响因子：
7.2
作者：
Meskauskiene, Rasa;Wuersch, Marco;Apel, Klaus
通讯作者：
Apel, Klaus
DOI：
10.1111/j.1432-1033.1996.0601p.x
发表时间：
1996-05-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Enriquez, JA;FernandezSilva, P;Montoya, J
通讯作者：
Montoya, J
DOI：
10.1073/pnas.0903086106
发表时间：
2009-06-09
影响因子：
11.1
作者：
Burzio, Veronica A.;Villota, Claudio;Burzio, Luis O.
通讯作者：
Burzio, Luis O.
影响因子：
14.9
作者：
Hyvärinen AK;Pohjoismäki JL;Reyes A;Wanrooij S;Yasukawa T;Karhunen PJ;Spelbrink JN;Holt IJ;Jacobs HT
通讯作者：
Jacobs HT