Inherent Specificity and Variation Sensitivity as Quantitative Metrics for RBP Binding

Interactions between RNAs and RNA binding proteins (RBPs) regulate gene expression in eukaryotic cells. RNA-RBP affinity measurements in vitro reveal diverse binding preferences, yet approaches to directly compare motif 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 variation sensitivity, which assesses tolerance to single nucleotide variations within a preferred motif. 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 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 to understand how inherent specificity and mutational sensitivity shape competitive RBP binding in ways not predicted by affinity alone. These models reveal that low-specificity RBPs can functionally enhance the specificity of high-specificity RBPs by occupying non-target sites. Overexpression of low-specificity RBM25 in cells recapitulates this effect, sharpening HNRNPC binding toward its preferred target motifs. Together, this work establishes inherent specificity and variation sensitivity as a quantitative basis for modeling how RBPs compete for RNA targets, complementing affinity-based descriptions of RNA-RBP interactions.