Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast Bact spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the Bact crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

目前学界对于剪接体（spliceosome）中，围绕pre-mRNA分支位点的内含子核苷酸与蛋白质之间的相互接触，以及剪接过程中这些接触的动态变化，所知寥寥。我们借助对在位点特异性标记的pre-mRNA上组装的纯化酵母Bact剪接体进行紫外线诱导交联，探究了蛋白质与pre-mRNA的相互作用，并利用纯化的剪接反应体系，分析了其在转化为催化激活的B*复合物与剪接第一步C复合物后的动态变化。研究检测到分支位点上下游的核苷酸与U2 SF3a/b蛋白Prp9、Prp11、Hsh49、Cus1及Hsh155之间存在相互接触，这表明此类相互作用在进化上具有保守性。此外，RES复合物蛋白Pml1与Bud13被证实可与分支位点下游的内含子区域产生接触。对比Bact复合物与B*、C复合物的交联图谱可见，U2与RES复合物蛋白与内含子的相互作用呈现动态变化特征。在剪接第一步催化反应发生后，我们观察到Cwc25与分支位点区域产生相互接触，且Prp8与Prp45与分支位点周围核苷酸的交联信号显著增强。Cwc25促进剪接第一步进行的活性并不依赖其与pre-mRNA的相互作用，这提示其通过蛋白质-蛋白质相互作用发挥调控功能。本研究为剪接体的蛋白质-pre-mRNA相互作用网络提供了重要的认知视角，并揭示了剪接体催化激活阶段与剪接第一步过程中全新的核糖核蛋白（RNP）重塑事件。