Strong Purifying Selection at Synonymous Sites in D. melanogaster

TL;DR

This study reveals that a significant portion of synonymous sites in D. melanogaster are under strong purifying selection, challenging the assumption that they are mostly neutral and highlighting their potential functional importance.

Contribution

It demonstrates that many synonymous sites are under strong selective constraint, and links this constraint to gene function and developmental stages, which is a novel insight.

Findings

22% of four-fold synonymous sites are under strong purifying selection

Strong constraint correlates with gene expression levels during development

Synonymous site constraint is independent of known functional elements like splicing enhancers

Abstract

Synonymous sites are generally assumed to be subject to weak selective constraint. For this reason, they are often neglected as a possible source of important functional variation. We use site frequency spectra from deep population sequencing data to show that, contrary to this expectation, 22% of four-fold synonymous (4D) sites in D. melanogaster evolve under very strong selective constraint while few, if any, appear to be under weak constraint. Linking polymorphism with divergence data, we further find that the fraction of synonymous sites exposed to strong purifying selection is higher for those positions that show slower evolution on the Drosophila phylogeny. The function underlying the inferred strong constraint appears to be separate from splicing enhancers, nucleosome positioning, and the translational optimization generating canonical codon bias. The fraction of synonymous sites under strong constraint within a gene correlates well with gene expression, particularly in the mid-late embryo, pupae, and adult developmental stages. Genes enriched in strongly constrained synonymous sites tend to be particularly functionally important and are often involved in key developmental pathways. Given that the observed widespread constraint acting on synonymous sites is likely not limited to Drosophila, the role of synonymous sites in genetic disease and adaptation should be reevaluated.