Abstract:
Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how U1 snRNP recognizes particular 5\' splice sites (5\'SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5\'SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5\'SS modulation. We have determined a sequential binding mechanism for modulation of -1A bulged 5\'SS 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 5\'SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5\'SS complexes. Branaplam is a ribonucleoprotein, not RNA duplex alone, targeting drug whose action depends on fundamental properties of 5\'SS recognition.
摘要:
剪接位点识别对于定义转录组至关重要。利沙普兰和兰纳普拉姆等药物改变了U1snRNP识别特定5'剪接位点(5'SS)的方式,并促进U1snRNP在这些位置的结合和剪接。尽管5种SS调节剂具有治疗潜力,它们相互作用和snRNP底物的复杂性排除了定义5'SS调制机制的可能性。我们已经结合了整体动力学测量和共定位单分子光谱学(CoSMoS),确定了通过branaplam调节-1A凸起的5'SS的顺序结合机制。我们的机制建立了U1-C蛋白与U1snRNP可逆结合,并且branaplam仅在与-1A凸出的5'SS接合后才与U1snRNP/U1-C复合物结合。结合和非结合的明确顺序解释了可逆的branaplam相互作用如何导致长寿命U1snRNP/5'SS复合物的形成。Branaplam是一种核糖核蛋白,不是单独的RNA双链体,靶向药物的作用取决于5'SS识别的基本性质。