Molecular Recognition During Pre-mRNA Splicing

总结 我们的项目的目标是了解指导前体mRNA剪接位点初始阶段的结构特征 识别，这是准确的前体mRNA剪接的关键，在人类疾病中经常失调。 我们集中在U2 AF，SF 1和SF 3B 1剪接因子指导U2小核核糖核蛋白（U2 snRNP）与前mRNA的3 '剪接位点连接。U2 AF亚基U2 AF 2和U2 AF 1识别 多嘧啶和AG-二核苷酸剪接位点信号。第三个亚基SF 1最初与分支结合 然后，前mRNA的点序列被U2 snRNP的SF 3B 1亚基置换。动态 SF 3B 1的磷酸化和去磷酸化是活性剪接体形成所必需的。在此以前， 我们在理解3 ′剪接位点初始步骤的分子基础方面取得了进展， 选择.使用X射线晶体学、生物物理学技术和功能测定， 在人类细胞中，我们已经表明U2 AF 2- RNA界面处的代表性癌症相关突变 破坏RNA结合和剪接。我们已经破译了结构细节，显示了U2 AF 2如何适应 剪接位点信号的不同核苷酸。通过互补单分子Förster共振能量转移 进一步揭示了U2 AF 2的构象随剪接位点的不同而发生变化 序列以及U2 AF 1亚基及其复发性癌症相关突变。我们建立 在人细胞中前体mRNA剪接过程中U2 AF 2与SF 1和SF 3B 1的重要功能界面， 发现磷酸化强烈降低了SF 3B 1与U2 AF 2的结合。然而，这些挑衅性的结果 提出了新的问题。首先，复发性U2 AF 2突变在神经发育障碍中的作用是什么？ 与癌症患者相比第二，动态SF 3B 1磷酸化和去磷酸化是如何 在剪接之前，U2 AF 2解离在时间上受到调节？第三，U2 AF 2、U2 AF 1和SF 1如何 亚基排列以准确识别剪接位点信号并确保剪接的保真度？我们解决 通过利用结构方法（包括X射线晶体学， 冷冻电子显微术、量热法和荧光法）和互补功能测定（包括共 免疫沉淀、前mRNA剪接测定和全转录组测序）。总的来说， 这些目标有助于理解3 ′剪接位点识别的结构和功能基础 以及它在人类疾病中的失调。

SUMMARY The goal of our project is to understand the structural features guiding the initial stages of pre-mRNA splice site recognition, which are critical for accurate pre-mRNA splicing and frequently dysregulated in human diseases. We focus on the U2AF, SF1 and SF3B1 splicing factors directing the U2 small nuclear ribonucleoprotein (U2 snRNP) to the 3´ splice sites of pre-mRNAs. The U2AF subunits, U2AF2 and U2AF1, recognize the polypyrimidine and AG-dinucleotide splice site signals. A third subunit, SF1 initially associates with the branch point sequence of the pre-mRNA then is displaced by the SF3B1 subunit of the U2 snRNP. Dynamic phosphorylation and dephosphorylation of SF3B1 is required for formation of the active spliceosome. Previously, we made progress towards understanding the molecular underpinnings of the initial steps of 3´ splice site selection. Using X-ray crystallography, biophysical techniques, and functional assays for pre-mRNA splicing in human cells, we have shown that representative cancer-associated mutations at the U2AF2 – RNA interface disrupt RNA binding and splicing. We have deciphered structural details showing how U2AF2 accommodates diverse nucleotides of splice site signals. By complementary single molecule Förster resonance energy transfer approaches, we further revealed that the U2AF2 conformation changes in response to different splice site sequences as well as the U2AF1 subunit and its recurrent cancer-associated mutation. We have established important, functional interfaces of U2AF2 with SF1 and SF3B1 during pre-mRNA splicing in human cells and discovered that phosphorylation strongly reduces SF3B1 binding to U2AF2. However, these provocative results raise new questions. First, what are the effects of recurrent U2AF2 mutations in neurodevelopmental disorders compared to those in cancers? Second, how is dynamic SF3B1 phosphorylation and dephosphorylation temporally regulated with U2AF2 dissociation prior to splicing? Third, how are the U2AF2, U2AF1, and SF1 subunits arranged to accurately recognize the splice site signals and ensure the fidelity of splicing? We address these questions in the aims of this proposal by leveraging structural approaches (including X-ray crystallography, cryoelectron microscopy, calorimetry, and fluorescence) and complementary functional assays (including co- immunoprecipitations, pre-mRNA splicing assays, and transcriptome-wide sequencing). Altogether, the results of these aims contribute to understanding the structural and functional underpinnings of 3´ splice site recognition and its dysregulation in human disease.