The structural effects of indel polymorphisms outside the binding site on RNA-protein interactions are shaped by selection.

Genomic variants influence phenotypes and organismal fitness, with their effects shaped by genomic context. In 3' untranslated regions, variants can alter phenotypes by influencing RNA-protein binding and subsequent post-transcriptional gene regulation. Here, we investigate how indel variants impact RNA-protein interactions from outside the binding site, through changes in RNA secondary structure. Our findings reveal that indels can significantly affect protein binding affinities over distances spanning tens of nucleotides, with longer indels exerting greater effects until saturation. Crucially, we find evidence that this effect is constrained by purifying selection. Naturally observed indel polymorphisms cause smaller changes in binding affinity than synthetic indels, and this signal of selection is more pronounced near HuR binding sites. We also find that the sequence context of HuR binding sites shows greater resilience to indel mutations compared to randomly selected sites. These results demonstrate that indel polymorphisms outside the binding site can modulate RNA-protein interactions through structural effects, and that purifying selection acts to filter out variants that disrupt critical interactions.