Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.原核基因组具有极高的信息压缩特性，通常可从同一基因座转录产生多种RNA分子。尽管反义RNA（antisense RNA）的研究已较为深入，但同链RNA中的内部RNA（intraRNAs）仍知之甚少。重叠阅读框的翻译究竟有多普遍，这一问题至今尚无定论。本研究以模式古菌盐沼盐杆菌（Halobacterium salinarum）为对象，旨在解答这一问题。我们针对盐沼盐杆菌NRC-1开展差异RNA测序（differential RNA-seq, dRNA-seq）分析，在部分内部转录起始位点（internal transcription start site, iTSS）中定位到内部RNA信号，并确定了与之对应的开放阅读框（open reading frame, ORF，即intraORFs）。通过构建C端标记蛋白，我们在kef1、acs3与orc4基因中，实验观测到了由内部RNA翻译精准预测的蛋白同工型。我们还从文献及质谱（mass spectrometry）数据库中检索到多例与内部RNA翻译相符的蛋白同工型，例如气囊蛋白基因gvpC1。我们在盐沼盐杆菌与大肠杆菌（Escherichia coli）的水平转移基因（horizontally transferred gene）中发现了内部RNA的存在证据，包括分子伴侣dnaK以及参与有氧呼吸的cydA基因。此外，在盐沼盐杆菌、大肠杆菌及酵母的通用延伸因子（elongation factor，aEF-2、fusA与eEF-2）相关基因中发现的内部RNA翻译证据，表明这是一种广泛存在于所有生命域的古老现象。