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 <i>locus</i>. 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 <i>Halobacterium salinarum</i>. In the present work we used differential RNA-seq (dRNA-seq) in <i>H. salinarum</i> 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 <i>kef1, acs3</i> and <i>orc4</i> 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 <i>gvpC1</i>. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone <i>dnaK</i> and the aerobic respiration related <i>cydA</i> in both <i>H. salinarum</i> and <i>Escherichia coli</i>. Also, intraRNA translation evidence in <i>H. salinarum, E. coli</i> and yeast of a universal elongation factor (<i>aEF-2, fusA</i> and <i>eEF</i>-2) suggests that this is an ancient phenomenon present in all domains of life.