PPubMed20 Dec 2012Cited 19×

On the Limitations of Using Ribosomal Genes as References for the Study of Codon Usage: A Rebuttal On the Use of Ribosomal Genes to Study Codon Usage

Ruth Hershberg, Dmitri A. Petrov, Ahmed Moustafa

Abstract

In a recent paper published in PLOS ONE, Wang et al. challenge our finding that the identity of optimal codons in different genomes follows a set of clear rules. Here we provide a rebuttal of their paper and demonstrate that the results of our original PLOS Genetics paper stand. This provides us with an opportunity to bring up an aspect of how codon usage has been studied that should be of general interest. The Wang et al. study, as well as many other studies, used ribosomal genes as a reference set for the study of patterns of codon usage. We discuss here the assumptions that are made in order to justify using ribosomal genes to study codon bias, suggest that this practice can at times be problematic, and discuss its limitations.

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Using ribosomal genes as a reference for codon usage can be misleading; the authors rebut Wang et al. and show genome‑wide codon patterns depend on GC content.

What’s new

i.Rebuttal of Wang et al. (2011) showing their favored‑codon results are incorrect
ii.Demonstration that ribosomal‑gene reference method can produce misleading codon‑bias conclusions
iii.Identification of the “going with the flow” trend linking favored codons to genomic GC content
iv.Quantitative analysis of how many non‑ribosomal genes are under selection across multiple bacterial genomes

How they did it

The authors applied the correlation method, identifying favored codons by tracking frequency changes in genes with increasing overall bias, and compared it to the ribosomal‑gene comparison method. They extracted the first 100 four‑fold and two‑fold degenerate codons from each gene (excluding ribosomal genes), created intergenic control coding segments (ICCS) by randomizing third positions, and measured codon bias using the Nc′ metric. Statistical tests (e.g., paired Mann‑Whitney) were used to assess differences between ribosomal and non‑ribosomal genes across genomes.

What they found

658/675 bacterial genomes show ribosomal genes over‑represented among the 100 most codon‑biased genes
In GC‑rich genomes (>50% intergenic GC), 94% have lower third‑position GC in ribosomal genes than in other genes (P<<0.0001, n=224)
GCT codon identified by Wang et al. occurs at 4.7% vs 3% in ribosomal vs non‑ribosomal genes, but averages only 8.7% vs 4.5% genome‑wide in GC‑rich bacteria
The number of non‑ribosomal genes under selection varies: majority in Chromobacterium violaceum, fewer in Bacillus subtilis, intermediate in Escherichia coli

Caveats

✕Assumes ribosomal genes are the only highly expressed genes under selection, which is often false
✕Assumes differences between ribosomal and non‑ribosomal codon usage stem solely from global selection, ignoring ribosomal‑specific constraints
✕The comparison method may miss favored codons when consistency across genes is lacking
✕Statistical significance does not guarantee biological relevance of rare codon enrichments

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