Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
BMC Genet. 2020 Mar 6;21(1):25. doi: 10.1186/s12863-020-0828-7.
POLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF.
Using PhyloCSF to survey alternative reading frames, we found a conserved coding signature in an alternative frame in exons 2 and 3 of POLG, herein referred to as ORF-Y that arose de novo in placental mammals. Using the synplot2 program, synonymous site conservation was found among mammals in the region of the POLG ORF that is overlapped by ORF-Y. Ribosome profiling data revealed that ORF-Y is translated and that initiation likely occurs at a CUG codon. Inspection of an alignment of mammalian sequences containing ORF-Y revealed that the CUG codon has a strong initiation context and that a well-conserved predicted RNA stem-loop begins 14 nucleotides downstream. Such features are associated with enhanced initiation at near-cognate non-AUG codons. Reanalysis of the Kim et al. (2014) draft human proteome dataset yielded two unique peptides that map unambiguously to ORF-Y. An additional conserved uORF, herein referred to as ORF-Z, was also found in exon 2 of POLG. Lastly, we surveyed Clinvar variants that are synonymous with respect to the POLG ORF and found that most of these variants cause amino acid changes in ORF-Y or ORF-Z.
We provide evidence for a novel coding sequence, ORF-Y, that overlaps the POLG ORF. Ribosome profiling and mass spectrometry data show that ORF-Y is expressed. PhyloCSF and synplot2 analysis show that ORF-Y is subject to strong purifying selection. An abundance of disease-correlated mutations that map to exons 2 and 3 of POLG but also affect ORF-Y provides potential clinical significance to this finding.
POLG 位于核染色体 15 上，编码 DNA 聚合酶 γ（Pol γ）。Pol γ 负责线粒体 DNA（mtDNA）的复制和修复。Pol γ 是大多数动物细胞中线粒体中唯一发现的 DNA 聚合酶。POLG 中的突变是线粒体疾病的最常见单基因原因，并已在 POLG ORF 的编码区中进行了映射。
使用 PhyloCSF 来调查备用阅读框，我们在 POLG 的外显子 2 和 3 中发现了备用框架中的保守编码特征，本文称之为 ORF-Y，它是在胎盘哺乳动物中从头出现的。使用 synplot2 程序，在 POLG 的 ORF 与 ORF-Y 重叠的区域中，在哺乳动物中发现了同义位点的保守性。核糖体谱数据显示，ORF-Y 被翻译，并且起始可能发生在 CUG 密码子上。对包含 ORF-Y 的哺乳动物序列的比对检查表明，CUG 密码子具有很强的起始上下文，并且在 14 个核苷酸下游开始形成一个保守的预测 RNA 茎环。这种特征与增强近同源非 AUG 密码子的起始有关。对 Kim 等人（2014 年）的人类蛋白质组草案数据集的重新分析产生了两个明确映射到 ORF-Y 的独特肽。还在外显子 2 中发现了另一个保守的 uORF，本文称之为 ORF-Z。最后，我们调查了与 POLG ORF 同义的 Clinvar 变体，发现这些变体中的大多数导致 ORF-Y 或 ORF-Z 中的氨基酸变化。
我们提供了一个新的编码序列 ORF-Y 的证据，该序列与 POLG ORF 重叠。核糖体谱和质谱数据表明 ORF-Y 是表达的。PhyloCSF 和 synplot2 分析表明，ORF-Y 受到强烈的纯化选择。大量与疾病相关的突变映射到 POLG 的外显子 2 和 3，但也影响 ORF-Y，这为这一发现提供了潜在的临床意义。
