Kondo Yasushi, Oubridge Chris, van Roon Anne-Marie M, Nagai Kiyoshi
Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Elife. 2015 Jan 2;4:e04986. doi: 10.7554/eLife.04986.
U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.
U1 小核核糖核蛋白颗粒（U1 snRNP）与前体信使核糖核酸（pre-mRNA）的 5' 外显子 - 内含子连接点结合，因此在 pre-mRNA 剪接的早期阶段发挥着关键作用。我们展示了工程化 U1 子结构的两种晶体结构，它们共同以原子分辨率揭示了 U1 snRNP 内几乎完整的蛋白质 - 蛋白质和 RNA - 蛋白质相互作用网络，并展示了 U1 snRNP 如何识别 pre-mRNA 的 5' 剪接位点。U1-C 的锌指与 pre-mRNA 和 U1 snRNA 的 5' 末端之间的双链体相互作用。RNA 双链体的结合通过 U1-C 与剪接连接点周围的 RNA 主链之间的氢键和静电相互作用得以稳定，但 U1-C 与 pre-mRNA 没有碱基特异性接触。该结构与 RNA 结合试验一起表明，U1 snRNP 对 5' 剪接位点核苷酸的选择主要通过与 U1 snRNA 的碱基配对实现，而 U1-C 则微调错配 5' 剪接位点的相对亲和力。
