Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Element Biosciences, San Diego, CA, USA.
Nat Commun. 2024 Oct 10;15(1):8776. doi: 10.1038/s41467-024-53124-5.
Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human 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 with 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 targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5'SS recognition.
剪接位点识别对于定义转录组至关重要。像利司扑兰和依替巴肽这样的药物改变了人类 U1 snRNP 识别特定 5'剪接位点（5'SS）的方式，并促进 U1 snRNP 在这些位置的结合和剪接。尽管 5'SS 调节剂具有治疗潜力，但它们相互作用的复杂性和 snRNP 底物的复杂性使得难以定义 5'SS 调节的机制。我们使用组合的集合动力学测量和共定位单分子光谱学（CoSMoS），确定了 branaplam 通过 -1A 膨出 5'SS 进行调节的顺序结合机制。我们的机制确立了 U1-C 蛋白可逆地结合到 U1 snRNP，并且 branaplam 仅在与 -1A 膨出 5'SS 结合后才结合到 U1 snRNP/U1-C 复合物。结合和去结合的强制性顺序解释了为什么可逆的 branaplam 相互作用会导致长寿命的 U1 snRNP/5'SS 复合物的形成。Branaplam 靶向核糖核蛋白，而不仅仅是 RNA 双链，其作用取决于 5'SS 识别的基本特性。
