Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
Mol Cell Biol. 2014 Jan;34(2):210-20. doi: 10.1128/MCB.00837-13. Epub 2013 Nov 4.
The yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP. We show here that Sad1 plays a role in maintaining a functional form of the tri-snRNP by promoting the association of U5 snRNP with U4/U6 di-snRNP. In the absence of Sad1, the U4/U6.U5 tri-snRNP dissociates into U5 and U4/U6 upon ATP hydrolysis and cannot bind to the spliceosome. The separated U4/U6 and U5 can reassociate upon incubation more favorably in the absence of ATP and in the presence of Sad1. Brr2 is responsible for mediating ATP-dependent dissociation of the tri-snRNP. Our results demonstrate a role of Sad1 in maintaining the integrity of the tri-snRNP by counteracting Brr2-mediated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP.
酵母 Sad1 蛋白先前在参与 U4/U6 二 snRNP 颗粒组装的因子筛选中被鉴定出来。Sad1 在体内和体外的前体 mRNA 剪接中都是必需的，其人类同源物已被证明与 U4/U6.U5 三 snRNP 相关联。我们在这里表明，Sad1 通过促进 U5 snRNP 与 U4/U6 二 snRNP 的结合，在维持三 snRNP 的功能形式方面发挥作用。在没有 Sad1 的情况下，三 snRNP 在 ATP 水解时会解离成 U5 和 U4/U6，并且不能与剪接体结合。在没有 ATP 的情况下，分离的 U4/U6 和 U5 在孵育时更有利于在 Sad1 的存在下重新结合。Brr2 负责介导三 snRNP 的 ATP 依赖性解离。我们的结果表明，Sad1 通过拮抗 Brr2 介导的三 snRNP 解离来维持三 snRNP 的完整性，并为三 snRNP 的动态平衡提供了见解。
