Inherent Specificity and Mutational Sensitivity as Quantitative Metrics for RBP Binding

Interactions between RNAs and RNA binding proteins (RBPs) regulate gene expression in eukaryotic cells. RNA-RBP affinities measured in vitro reveal diverse binding specificities, yet approaches to directly compare specificities across RBPs are lacking. Here, we introduce two quantitative metrics: inherent specificity, which measures how selectively an RBP distinguishes its strongest binding motif from all possible motifs, and mutational sensitivity, which assesses tolerance to single nucleotide variations within preferred motifs. Analyzing high-throughput sequencing datasets, we compared these metrics across 100 RBPs in vitro and 27 RBPs in cells, finding a strong correlation between in vitro and cellular measurements for RBPs that bind RNA independently of a local structural context. Through RNA binding domain swap CLIP experiments between low-specificity RBM25 and high-specificity HNRNPC, we demonstrated that sequence specificity can be measurably transferred between protein contexts. Using these insights, we developed mathematical models illustrating how inherent specificity and mutational sensitivity influence competitive RBP binding, observing that low-specificity RBPs can functionally enhance the specificity of high-specificity RBPs by occupying non-target sites. Together, our results provide a quantitative framework for modeling RNA-RBP interactions and designing RBPs with targeted specificity.