Rearrangements within the U6 snRNA core during the transition between the two catalytic steps of splicing. Eysmont et al.

The RNA catalytic core of spliceosomes as visualized by cryo-EM remains unchanged at different stages of splicing. However, we demonstrate that mutations within the core of yeast U6 snRNA modulate conformational changes between the two catalytic steps. We propose that the intramolecular stem-loop (ISL) of U6 exists in two competing states, changing between a default, non-catalytic conformation and a transient, catalytic conformation. Whereas stable interactions in the catalytic triplex promote catalysis and their disruptions favor exit from the catalytic conformation, destabilization of the lower ISL stem promotes catalysis and its stabilization supports exit from the catalytic conformation. Thus, in addition to the catalytic triplex, U6-ISL acts as an important dynamic component of the catalytic center. The relative flexibility of the lower U6-ISL stem is conserved across eukaryotes. Similar features are found in U6atac and domain V of group II introns, arguing for the generality of the proposed mechanism.通过冷冻电子显微镜（cryo-EM）观测得到的剪接体RNA催化核心，在剪接的不同阶段均保持稳定。然而，本研究证实，酵母U6小核RNA（small nuclear RNA, snRNA）核心区域的突变可调控两个催化步骤之间的构象变化。我们提出，U6的分子内茎环（intramolecular stem-loop, ISL）存在两种竞争性构象状态，可在默认的非催化构象与瞬时的催化构象之间相互转换。催化三链体的稳定相互作用可促进催化反应，其解离则会促使构象脱离催化状态；而U6-ISL下部茎的去稳定化可促进催化反应，其稳定化则会辅助构象退出催化状态。因此，除催化三链体外，U6-ISL亦是催化中心的重要动态组成部分。U6-ISL下部茎的相对灵活性在真核生物中具有保守性。II型内含子（group II introns）的U6atac与V结构域亦存在类似特征，这佐证了本研究提出的机制具有普适性。