Shadow ORFs illuminated: long overlapping genes inPseudomonas aeruginosaare translated and under purifying selection

Abstract

The existence of overlapping genes (OLGs) with significant coding overlaps revolutionises our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the medically important Gram-negative bacterium Pseudomonas aeruginosa. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing data. Translation of both OLGs was confirmed by ribosome profiling and mass spectrometry. Quantitative proteomics of samples taken during different phases of growth revealed regulation of protein abundances, implying biological functionality. Both OLGs are taxonomically highly restricted, and likely arose by overprinting within the genus. Evidence for purifying selection further supports functionality. The OLGs reported here are the longest yet proposed in prokaryotes and are among the best attested in terms of translation and evolutionary constraint. These results highlight a potentially large unexplored dimension of prokaryotic genomes.

Overlapping genes in natural and engineered genomes

“…The unusual sequence characteristics of long overlapping CDSs may have also contributed to the difficulty of their discovery, resulting in undercounting 40 , 41 . One reason put forward to explain why long nested overlaps should be rare includes the evolutionary burden of maintaining larger overlaps, although evidence to the contrary showing positive selection at overlaps 27 , 42 , 43 shows that this explanation may be too simplistic. Selection for long convergent overlaps has been shown to have a strong reading frame bias and it has been suggested that retention involves positive selection at the birth of the overlap, followed by purifying selection afterwards 27 .…”

“…Selection for long convergent overlaps has been shown to have a strong reading frame bias and it has been suggested that retention involves positive selection at the birth of the overlap, followed by purifying selection afterwards 27 . Recently, an overlapping protein-encoding CDS with extensive 603 bp overlap has been discovered embedded in the highly conserved ompA gene in enterohaemorrhagic Escherichia coli 44 , showing that, with improved measurement tools, more of these long nested overlaps may be discovered 42 .…”

Three Novel Antisense Overlapping Genes in E. coli O157:H7 EDL933

“…An apparent scenario would be an emergence through overprinting ( 63 ) after the split of the Escherichia / Shigella clade. Interestingly, many of recently characterized overlapping genes are taxonomically restricted and were suggested being orphans ( 21 , 22 , 24 , 25 ). Orphans presumably code for non-essential, specialized functions that benefit the particular organism.…”

“…In recent years, an increasing number of studies provided strong indications for the existence of long overlapping genes in bacteria ( 10 , 19 , 20 ). In addition, evidence for the functionality of non-trivial overlapping genes in bacteria is accumulating ( 3 , 21 – 25 ).…”

Transcriptional analysis in bacteriophage Fc02 of Pseudomonas aeruginosa revealed two overlapping genes with exclusion activity

“…However, frequently the evidence provided is partial, the function of one or both overlapped genes is unknown, or is not clear whether the corresponding mRNA is transcribed and translated into protein. More frequently, the regulation regime of the proteins expressed and their functions are unknown (Kreitmeier et al, 2022). The historic case of overlapped genes in phage ϕX174 (Sanger et al, 1977) has been updated recently.…”