Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA 3′-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of Saccharomyces cerevisiae spliceosomes in vitro. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both ( B Lsm + NTC Δ U4 ) and provide a kinetic framework for its formation and transformation during activation. Prior to B Lsm + NTC Δ U4 assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing-dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. We propose that this interaction may play a role in formation of transient spliceosome intermediates formed during activation.

中文翻译：

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单分子荧光显微镜鉴定剪接体激活过程中的瞬时中间体

剪接体激活是为 RNA 剪接创建催化位点的过程，并在剪接体组装后在每个内含子上重新发生。数十种因子与激活剪接体结合或从中释放，包括与 U6 小核 RNA 3'-末端结合的 Lsm2-8 异七聚体环。必须释放 Lsm2-8 以允许包含 Prp19 的复合体（NineTeen Complex，NTC）稳定活性位点；然而，人们对导致 Lsm2-8 发布之前和之后的事件和动态交互的时间顺序知之甚少。我们使用共定位单分子光谱 (CoSMoS) 来可视化激活过程中的 Lsm2-8 动力学酿酒酵母体外剪接体。Lsm2-8 被招募为 tri-snRNP 的一个组成部分，并在包含 Prp19 的复合物 (NTC) 整合后释放。尽管 Lsm2-8 和 NTC 在酵母 B 复合体剪接体的现有低温电镜结构中相互排斥，但我们确定了一个包含两者的瞬时中间体（ 乙 最小值 + 负温度系数 △ U4 ) 并为其在激活过程中的形成和转化提供动力学框架。之前 乙 最小值 + 负温度系数 △ U4 组装后，NTC 快速且可逆地对剪接体进行采样，这表明一种机制可以防止 NTC 被无法正确激活的有缺陷的剪接体隔离。在互补合奏分析中，我们表明可以在 Lsm2-8 与 U2 和 U6 螺旋 II RNA 之间形成依赖于碱基配对的三元复合物。我们提出这种相互作用可能在激活过程中形成的瞬时剪接体中间体的形成中发挥作用。