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

## TL;DR

Indel polymorphisms outside RNA binding sites can affect protein binding through structural changes, and natural selection limits disruptive variants.

## Contribution

Shows that indels influence RNA-protein interactions via structural effects and are filtered by purifying selection.

## Key findings

- Indels can alter RNA-protein binding affinities over tens of nucleotides, with larger indels having stronger effects.
- Naturally occurring indels cause smaller binding changes than synthetic ones, indicating purifying selection.
- HuR binding sites show greater resilience to indel mutations compared to random sites.

## Abstract

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.

Genetic differences between individuals can influence how genes are regulated, leading to changes in health outcomes. While variants called indels are known to influence regulatory proteins by altering their direct binding sites, our study investigates how these variants affect RNA-protein interactions from outside the binding site by altering the structure of RNA molecules. We discovered that indels far from protein binding sites can significantly weaken or strengthen binding, with larger indels having a bigger impact. Interestingly, our analysis shows that natural selection works to minimize such disruptions: indel polymorphisms that greatly disturb RNA-protein binding are less likely to persist in populations. Moreover, protein binding sites near key regulatory regions, such as those bound by the HuR protein, are more resilient to the effects of indels. These findings highlight how genetic changes can influence gene regulation indirectly and show that the genome is shaped to protect critical regulatory mechanisms from such disruptions. This work sheds light on the interplay between genetic variation, RNA structure, and gene regulation, advancing our understanding of how genetic changes impact health and evolution.