Codon bias shapes bacterial small RNA binding sites within protein-coding sequences

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with target mRNAs, affecting their stability and translation. While sRNA binding sites were initially identified in 5’ untranslated regions of mRNAs, consistent with their role as translation-initiation regulators, recent large-scale studies have revealed sRNA binding sites within protein-coding sequences, suggesting additional regulatory mechanisms. It is intriguing to explore how the latter sRNA binding sites are adjusted with the reading frame and what selection forces maintain them within the coding sequence through evolution. Using RIL-seq data, we determined prime sRNA binding positions within coding sequences, which are positions within the inferred binding-site motif that show exceptionally high conservation across target sequences (≥95%), indicating their putative importance for sRNA–mRNA base-pairing. We found that these positions are mostly adjusted with the reading frame and correspond to the most frequent codons, high above random expectation. This suggests that frequent codons may facilitate sRNA–mRNA encounters and that codon usage bias influences binding site formation via selective pressures. Conservation analysis across genomes in the Enterobacterales order revealed that prime positions show relatively high conservation of base-pairing interactions. However, in some genomes base-pairing in these positions may be hampered due to the degeneracy of the genetic code. This is often compensated for by other positions that conserve the base-pairing interactions, ensuring the maintenance of a requisite number of base pairs for sustaining the sRNA–target interaction. Our findings highlight the importance of distinct interacting positions as well as an adequate number of base pairs for sustaining sRNA–target interactions.