运用液体核磁共振研究人类mRNA加工因子Fip1调控CPSF识别多聚腺苷酸化信号的分子基础

In eukaryotic organisms most messenger RNA precursors must undergo cleavage and polyadenylation at their 3′-end for maturation, called 3′-end processing. Alternative polyadenylation that occurs during this process has emerged as a fundamental mediator of gene expression. It has recently been shown that malignant transformation is associated with mRNAs containing significantly shorter 3’-UTRs produced by alternative polyadenylation. Despite its obvious biological importance, the structural biology of 3’-end processing has remained largely unexplored. As one of the most important mRNA processing factors, Fip1 plays direct role in linking cleavage factor Im (CFIm) and cleavage and polyadenylation specificity factor (CPSF). It has recently been identified as a critical regulator of the global alternative polyadenylation profile. The goal of this project is to study the structural basis for the formation of Fip1-CPSF30 complex by solution NMR, unveil how Fip1 and CPSF30 recognize each other at the atomic level, and determine if the structural module of Fip1-CPSF30 is independent of or spatially restricted to the core CPSF complex. Furthermore, we will integrate the structural module of Fip1-CPSF30 into the core CPSF complex by molecular docking and MD simulation, and draw a picture showing how Fip1 regulates PAS RNA recognition by the CPSF complex. This project would provide fundamental structural insights into how Fip1 is involved in the pre-mRNA’s poly(A) site selection during mRNA 3′-end processing.

真核生物的mRNA大都要经历3'末端切割和多聚腺苷酸化的成熟过程，称之为3'末端加工。在这个水平发生的由可变多聚腺苷酸化介导的基因调控影响着细胞分化与增殖，与肿瘤形成密切相关。尽管如此，我们对参与其中的蛋白机器的结构和功能的了解远远不够。Fip1是关键的mRNA加工因子，负责连接剪切因子CFIm与剪切和多聚腺苷酸化特异性因子CPSF、参与polyA位点识别和可变多聚腺苷酸化调控。本项目旨在运用液体核磁共振方法解析Fip1与CPSF30复合物的溶液结构、分析Fip1与CPSF30的相互作用机制、阐明Fip1与CPSF30作用模块相对于CPSF复合物主体的动力学特性。基于以上结果，结合分子动力学模拟手段，探讨Fip1如何调控CPSF识别polyA位点。该项目为研究mRNA前体3'末端加工的分子机制，尤其是Fip1在polyA信号识别及在可变多聚腺苷酸化的调控中如何发挥作用提供了分子基础。

CPSF蛋白复合物是重要的mRNA前体3'末端加工机器，负责对PAS信号的识别和连接其他mRNA前体加工因子。即使CPSF核心复合物与PAS结合的结构已经得到解析，这个蛋白机器是如何完成对PAS信号识别的分子机制依然不清楚。并且，其中较为受关注的与APA密切相关的蛋白因子Fip1，到底在CPSF识别PAS过程中扮演何种角色也是未知。本研究运用前沿液体核磁共振技术解析了Fip1-CPSF30复合物的溶液结构，确定了这部分模块在CPSF核心复合物中独立的空间构象，还全面阐释了CPSF不同亚基协同识别PAS RNA的动态过程。我们通过拆解CPSF复合物中关键的PAS结合蛋白，分别研究他们对RNA识别的特性，揭示了CPSF各亚基协同识别PAS的分子机制：CPSF30的锌指结构域ZnF23负责检索富含AA的RNA序列；Fip1-ZnF45调节ZnF23与AA结合强度，潜在参与APA位点的选择；Wdr33通过提供底物RNA折叠的构象熵来极大的增强了CPSF与PAS RNA的结合强度，完成了CPSF对PAS的特异性识别。该研究在广义层面为复合物蛋白机器识别mRNA信号分子机制的研究提供了新的视角，在狭义层面为CPSF复合物在APA位点选择的分子机制上提供了重要的启示。

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