A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression
A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression
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DOI：
10.1093/hmg/ddu352
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发表时间：
2014-12-01
影响因子：
3.5
通讯作者：
Lightowlers, Robert N.
中科院分区：
文献类型：
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作者：
Wilson, William C.;Hue-Tran Hornig-Do;Lightowlers, Robert N.
人类线粒体多聚腺苷酸聚合酶（mtPAP）中的p.N478D错义突变先前被认为与痉挛性共济失调伴视神经萎缩有关。在这项研究中，我们研究了从家庭成员建立成纤维细胞系。纯合突变导致评估的所有线粒体转录物的多聚腺苷酸化的损失;然而，寡聚腺苷酸化被保留。有趣的是，这对转录物稳定性有不同的影响，这取决于特定的转录物种类。这些变化伴随着氧化磷酸化复合物I和IV的严重损失，以及从头线粒体蛋白质合成的扰动。转录多聚腺苷酸化和呼吸链复合物的减少被野生型mtPAP的过表达有效地挽救。突变型和野生型mtPAP都定位于线粒体RNA加工颗粒，从而消除了作为缺陷性聚腺苷酸化原因的错误定位。体外多聚腺苷酸化测定显示，突变蛋白质的活性严重受损，其在RNA底物上仅产生短的oligo（A）延伸，而与RNA二级结构无关。添加LRPPRC/SLIRP（一种线粒体RNA结合复合物）增强了野生型mtPAP的活性，导致总尾长增加。LRPPRC/SLIRP效应尽管存在，但在突变的mtPAP中不太明显，与RNA二级结构无关。我们的结论是：（i）mtPAP的聚合酶活性可以通过LRPPRC/SLIRP的存在进行调节，（ii）N478 D mtPAP突变降低聚合酶活性，（iii）poly（A）长度的改变足以引起转录后表达的失调和呼吸链复合物的致病性缺乏。
The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.