Thermodynamic Characteristics of pre-mRNA Splice Site Recognition

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Here, we reveal that an unusually large enthalpy-entropy compensation underlies recognition of polypyrimidine (Py) splice site signals. Competitive binding to Py tract splice site signals represents a prevalent means for alternative pre-mRNA splicing. The thermodynamic forces driving association of splicing factors with single-stranded (ss) pre-mRNAs represents a gap in the current understanding of splice site selection. We compared Py tract interactions among three splicing factors: (1) U2AF65, an essential pre-mRNA splicing factor that recognizes constitutive 3′ splice site signals; (2) Sex-Lethal, a prototypical alternative splicing factor that antagonizes U2AF65; and (3) TIA-1, an alternative splicing factor that promotes use of specific 5′ splice sites. All three proteins bound polyuridine (U20) sequences with comparable or higher affinity than natural splice site sequences in fluorescence anisotropy assays. Consistent with the ability of Sex-Lethal to outcompete U2AF65 during splice site selection, U2AF65 displayed the lowest and Sex-Lethal the highest affinities for the RNA sites.The enthalpic and entropic contributions were investigated in detail using ITC, initially using the homogeneous U20 site to avoid sequence-dependent complications of the binding isotherms. All three splicing factors exhibited an unusually large enthalpy-entropy compensation underlying U20 binding, with magnitudes ∼10-fold greater than those of typical protein-protein or protein-ligand complexes. Given that full thermodynamic characterizations of protein association with single-stranded RNAs are rare, this raised the question of the source of this unusual thermodynamic signature: Is a large enthalpy-entropy compensation a general characteristic of ssRNA binding, an inherent property of Py tracts, or a signature of sequence-specific ssRNA recognition? These possibilities are clarified by thermodynamic comparison of purine-tract association by these Py tract splicing factors, contrasted with the purine-specific protein PAB.