Are Antisense Proteins in Prokaryotes Functional?

Abstract

Many prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits further research, including insights from studies of molecular interactions and detailed evolutionary analysis. There are multiple lines of evidence, however, that many of these newly discovered proteins are of use to the organism. Condition-specific phenotypes have been characterized for a few. These proteins should be added to genome annotations, and the methods for predicting them standardized. Evolutionary analysis of these typically young sequences also may provide important insights into gene evolution. This research should be prioritized for its exciting potential to uncover large numbers of novel proteins with extremely diverse potential practical uses, including applications in synthetic biology and responding to pathogens.

Evidence for Numerous Embedded Antisense Overlapping Genes in DiverseE. coliStrains

“…The naming “conflicts” exhibits how scientists have perceived OLGs (Venter et al, 2011); however, it is good and necessary that such ‘unfitting’ data are reported. Further examples of studies showing evidence for antisense genes with overlapping ORFs in prokaryotes have been recently reviewed (Ardern et al, 2020). In addition, we argue that the recognition of overlapping ORFs using recent annotation pipelines strongly indicates protein coding.…”

“…Here, we focus on antisense overlaps as they can be detected in ribosome profiling data. The potential widespread functionality of such overlapping ORFs has recently been proposed as a hypothesis to investigate, in light of evidence across diverse bacteria (Ardern et al, 2020). However, only very few cases of such gene pairs, in which their ORFs overlap extensively, have been characterized phenotypically in E. coli (Fellner et al, 2014;Fellner et al, 2015;Haycocks and Grainger, 2016;Hücker et al, 2018a;Hücker et al, 2018b;Vanderhaeghen et al, 2018;Zehentner et al, 2020) in strong contrast to more than 128.000 non-overlapping genes known in the E. coli pan-genome (Park et al, 2019).…”

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

“…Verifying OLGs using mass spectrometry (MS) is difficult because most OLGs appear to be short and weakly expressed, and proteomics has limited abilities in detecting such proteins 22 . RNA sequencing led to the discovery of many antisense transcripts, but whether many of these are translated is controversial 23 . More recently, mRNA protected by ribosomes after enzymatic degradation has been sequenced using “ribosome profiling” 24 (RiboSeq), showing evidence of translation for antisense transcripts 16,23 .…”

“…RNA sequencing led to the discovery of many antisense transcripts, but whether many of these are translated is controversial 23 . More recently, mRNA protected by ribosomes after enzymatic degradation has been sequenced using “ribosome profiling” 24 (RiboSeq), showing evidence of translation for antisense transcripts 16,23 . However, artefacts may occur due to structured RNAs 25 .…”

Computational design of genes encoding completely overlapping protein domains: Influence of genetic code and taxonomic rank

“…While a mutation in a stop codon can easily create a short, trivial overlap in neighbouring genes as a chance event, longer, non-trivial overlaps should only be maintained in a genome if the overlapping region encodes a part of the protein essential for its function for both genes. There are a few hypothetical reasons why genes might overlap, and the evidence for functional antisense overlaps in prokaryotes has been discussed in a recent review [7]. While the reduction of genome size is particularly relevant only for some viruses [8,9], it has also been studied in bacteria [10].…”