Quantifying evolution of SNPs that affect RNA secondary structure in Arabidopsis thaliana genes

Single-stranded RNA molecules have the ability to form intramolecular bonds between nucleotides and create secondary structures. These structures can have phenotypic effects, meaning mutations that alter secondary structure may be subject to natural selection. Here we examined the population genetics of these mutations within Arabidopsis thaliana genes. We began by identifying derived SNPs with the potential to alter secondary structures within coding regions, using a combination of computational prediction and empirical data analysis. We then examined nucleotide diversity and allele frequencies of these “pair-changing mutations” (pcM) in 1,001 A. thaliana genomes to infer selective pressures on these sites. The pcM SNPs at synonymous sites had an 8.8% reduction in nucleotide diversity relative to non-pcM SNPs at synonymous sites, and they were found at lower allele frequencies. We used demographic modeling to estimate selection coefficients from the pcM allele frequency spectrum, finding that pcMs had estimated selection coefficients ∼3 to 5x smaller than nonsynonymous mutations. Additionally, we explored associations of pcMs with bioclimatic variables and gene expression. They exist at higher frequencies in higher latitudes and colder environments, with mean annual temperature explaining 51% of the variation in pcM frequency among subpopulations. Alleles containing pcMs had an average reduction of 137.4 normalized counts compared to genes with the ancestral secondary structure (mean expression = 3215.7 normalized counts). Overall, we conclude that structure-altering mutations are subject to intermediate levels of selection.