Donovan Benjamin T, Wang Bixuan, Garcia Gloria R, Mount Stephen M, Larson Daniel R
Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
bioRxiv. 2024 Jul 13:2024.07.12.603320. doi: 10.1101/2024.07.12.603320.
The spliceosome is a megadalton protein-RNA complex which removes introns from pre-mRNA, yet the dynamic early assembly steps have not been structurally resolved. Specifically, how the spliceosome selects the correct 3' splice site (3'SS) amongst highly similar non-functional sites is not known. Here, we develop a kinetic model of splice site selection based on single-molecule U2AF heterodimer imaging and . The model successfully predicts alternative splicing patterns and indicates that 3'SS selection occurs while U2AF is in complex with the spliceosome, not during initial binding. This finding indicates the spliceosome operates in a 'partial' kinetic proofreading regime, catalyzed in part by the helicase DDX42, which increases selectivity to the underlying U2AF binding site while still allowing for efficient forward progression.
剪接体是一种兆道尔顿级别的蛋白质-RNA复合物，可从前体mRNA中去除内含子，但动态的早期组装步骤尚未在结构上得到解析。具体而言，剪接体如何在高度相似的无功能位点中选择正确的3'剪接位点（3'SS）尚不清楚。在此，我们基于单分子U2AF异源二聚体成像开发了一种剪接位点选择的动力学模型。该模型成功预测了可变剪接模式，并表明3'SS选择发生在U2AF与剪接体形成复合物时，而非初始结合期间。这一发现表明剪接体在一种“部分”动力学校对机制下运行，部分由解旋酶DDX42催化，这增加了对潜在U2AF结合位点的选择性，同时仍允许高效的正向进程。
