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

Abstract

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.

Keywords: Halobacterium salinarum; IntraRNA; LUCA; aEF-2; acs3; alternative protein; alternative transcript; ancient phenomenon; archaea; cydA; dRNA-seq; dRNAseq; differential RNA-seq; dnaK; fusA; gvpC; internal RNA; intraORF; intraORFeome; isoform; kef1; orc4; overlapping coding RNA; protein isoforms; uORF.

Figure 1.

Sequence composition of regions upstream of genes and intraRNAs. Logo representation of upstream regions of gTSS (A) and iTSS (B). Relative positions are shown positioning TSS as zero.

Figure 2.

intraRNA translation validation. Western blots for chromosomally tagged acs3 (A), kef1 (B) and orc4 (D). # indicates the full length protein and * the isoform translated from the intraRNA. Panels on the right (A and B) show the gene organization with PFAM domains, iTSS (green arrow) and the putative start codon for the protein isoform. (C) Aligned reads coverage along genomic coordinates for TEX+ (dark green) and TEX- (green) (arbitrarily scaled and normalized). Coding sequence is in reverse strand (orange rectangle, locus ID inside, 5ʹ→3ʹ direction is right to left). Forward and reverse coverage signals are shown above and below horizontal axis respectively. Domain annotation (blue rectangle), identified iTSS (green triangle), all possible archaeal start codons (green) and stop codon (red) are shown. Predicted TIS is highlighted (magenta). (D) Western blot of C-terminally FLAG-tagged Orc4 protein (#) and isoform (*).