A Sequential Binding Mechanism for 5' Splice Site Recognition and Modulation for the Human U1 snRNP
Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how U1 snRNP recognizes particular 5 splice sites (5SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5SS modulation. We have determined a sequential binding mechanism for modulation of -1A bulged 5SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged a -1A bulged 5SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5SS complexes. Branaplam is a ribonucleoprotein, not RNA duplex alone, targeting drug whose action depends on fundamental properties of 5SS recognition.