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

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Bacterial small RNAs (sRNAs) are key regulators of gene expression, primarily through base pairing with target mRNAs.
• While initially studied in 5' untranslated regions, sRNA binding sites are now recognized within protein-coding sequences, indicating novel regulatory roles.
• The evolutionary maintenance and frameshifting of these intragenic sRNA binding sites remain poorly understood.

Purpose of the Study:

• To investigate the evolutionary pressures and frameshifting mechanisms governing bacterial small RNA binding sites within coding sequences.
• To identify conserved, functionally important positions within these intragenic sRNA binding sites.
• To explore the interplay between codon usage bias, evolutionary conservation, and sRNA-mRNA interactions.

Main Methods:

• Utilized RIL-seq data to identify prime sRNA binding positions within coding sequences, defined by high conservation (≥95%) within binding site motifs.
• Analyzed the relationship between prime binding positions, reading frame, and codon usage frequency.
• Performed cross-genome conservation analysis across Enterobacterales to assess the maintenance of base pairing interactions.

Main Results:

• Prime sRNA binding positions within coding sequences were found to align with the reading frame and correspond to frequently used codons, exceeding random expectations.
• Codon usage bias and evolutionary pressures appear to influence the establishment and maintenance of these intragenic binding sites.
• While some positions showed conservation of base pairing, degeneracy of the genetic code sometimes necessitated compensation by other positions to maintain sufficient base pairing interactions.

Conclusions:

• Intragenic sRNA binding sites are shaped by evolutionary selection, favoring frequent codons to enhance sRNA-mRNA encounter probability.
• The maintenance of sRNA-target interactions relies on a combination of conserved interacting positions and sufficient overall base pairing, despite challenges posed by genetic code degeneracy.
• Distinct interacting positions and an adequate number of base pairs are crucial for sustaining functional sRNA-target interactions within bacterial coding sequences.