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.

Keywords: antisense transcription; antisense translation; function; gene annotation; overlapping gene; selected effects.

FIGURE 1

Reported potential protein-coding ORFs overlapping in antisense, based on ribosome profiling experiments. (A) Reported antisense OLGs in E. coli K12 (NC_000913.3) – Weaver et al. (2019), Friedman et al. (2017; sRNAs with evidence of translation). (B) Reported antisense OLGs in M. tuberculosis (NC_000962.3) –Smith et al. (2019). Annotated genes gray. Antisense blue.

FIGURE 2

Expression and regulation of antisense genes as demonstrated by ribosome profiling experiments; aligned ribosome protected fragments shown as reads per million at each site after removal of rRNA and tRNA reads. (A) laoB gene in E. coli O157:H7 Sakai; expression in LB medium is higher than in BHI. (B) ano gene in E. coli O157:H7 Sakai; expression in LB medium versus BHI is constant. (C) yidD (MW1733) gene, part of non-contiguous (antisense) operon, in Staphylococcus aureus. Expression in positive and negative strands is positively correlated – high in the absence of the antibiotic azithromycin, low when it is added.

FIGURE 3

A subset of embedded antisense ORFs are well conserved in the genomes situated taxonomically between E. coli and Citrobacter rodentium. (A) Phylogenetic tree showing 13 representative genomes used, derived from the genome taxonomy database (GTDB). (B) Median pairwise amino acid identity and similarity (gray) among orthologs for 1000 annotated genes with single copy orthologs in all of the 13 genomes. As a comparison, the effect on identity and similarity of adding random mutations to simulated sequences is shown (orange). There is a clear bias toward variants which result in higher “similarity.” (C) Conservation of antisense embedded ORFs (blue), as compared to the identity-similarity relationship observed for control randomly mutated sequences (red) simulated as before but translated in antisense. Many antisense embedded ORFs are highly conserved and a subset also shows a bias toward similarity.