PTBP1介导的RNA高级结构动态变化调控可变剪接的机制研究

Polypyrimidine Tract-Binding Protein 1 (PTBP1) is a master regulator of cell development and differentiation. It is usually overexpressed in diverse cancer cells. We previously mapped the genome-wide binding sites of PTBP1 and constructed a functional RNA map based on position related splicing outcome. The results showed that PTBP1 can function as either splicing repressor or enhancer by binding to different positions in the pre-mRNA. However, the underling mechanism is still obscure. Since PTBP1 can function as dimer in vivo via 23-Cys (C23), we thus speculate that some distant PTBP1 binding sites may be close to each other via PTBP1 dimerization, which will change the higher order structure of pre-mRNA and cause the alternative proximity of distal splicing signal. Such dynamic regulation of RNA structure may contribute to PTBP1 mediated positional effects in alternative splicing. To test this hypothesis, we have made PTBP1 C23S isogenic HeLa cell lines and identified the binding sites for both monomer and dimer. With these cell lines, we plan to perform paired-end RNA sequencing to identify the splicing events regulated by PTBP1 dimerization. To further unravel PTBP1 mediated long-rang RNA interactions, we will adopt RBP-3C (RNA binding protein mediated contacts and conformation capture) technique, which was recently developed in our lab and can be used to map RNA in situ higher order structure, to identify PTBP1 regulated RNA-RNA interaction sites. Finally, by combining RNA-seq, CLIP-seq and PTBP1-3C data together, we hope to elucidate the mechanism of PTBP1 dimerization mediated long-distance RNA interactions in alternative splicing regulation. Furthermore, we will develop neural network models to systematically identify RNA tertiary structures mediated splicing events. This proposal not only represents the first example of systematically studying the RNA tertiary structures in alternative splicing, but also lay the foundation for understanding the pathological mechanisms of cervical cancer and glioma from a new angle.

RNA结合蛋白PTBP1是细胞发育和分化的关键调节因子，在癌细胞中异常高表达。我们前期绘制了PTBP1调控可变剪接的RNA图谱，发现了它促进或抑制可变剪接的位置效应，但具体分子机制不明。PTBP1在体内可借助第23位半胱氨酸（C23）形成二聚体，我们推测PTBP1有可能通过二聚化将远距离的RNA结合位点拉近，进而影响剪接信号的可变选择。为了验证该假说，我们构建了C23S突变型细胞系，鉴定了PTBP1单体和二聚体的结合位点。在此基础上，本申请将通过RNA-seq鉴定PTBP1二聚体调控的可变剪接事件，并利用新近开发的RBP-3C方法探寻二聚化介导的RNA高级结构集。通过综合分析RNA-seq、CLIP-seq和RBP-3C数据，阐明PTBP1二聚化在可变剪接调控中的作用机制，并发展神经网络预测与RNA高级结构相关的可变剪接事件，从RNA高级结构的角度解释宫颈癌和神经胶质瘤等癌症发生的机理。

在可变剪接调控过程中，PTBP1蛋白能够结合在pre-mRNA不同位置，以促进或者抑制可变外显子的剪接选择。在细胞内，PTBP1可借助第23位半胱氨酸（Cys23，C23）形成二聚体，我们推测PTBP1有可能通过二聚化将远距离的RNA结合片段拉近形成空间结构，进而调控可变外显子的剪接过程。为了验证这一假设并深入阐明前述位置效应背后的分子机制，我们首先开发了一种CRIC-seq新技术以捕获特定蛋白质介导形成的RNA-RNA原位空间互作信息。以HeLa细胞为研究对象，我们利用这一技术解析了PTBP1蛋白介导的RNA空间互作图谱，并据此发现PTBP1能够通过二聚化介导可变外显子两侧内含子内部形成空间结构以增强内含子的剪除速度进而促进可变外显子的选择，而介导两侧内含子之间形成空间结构以抑制可变外显子的剪接选择。这种“位置效应规则”能够用于预测PTBP1对可变剪接的调控方向性。进一步，我们揭示，肿瘤样本中的非编码突变位点能够破坏PTBP1蛋白与RNA的结合、影响RNA结构的形成，从而引起剪接异常，最终改变肿瘤的发生发展过程。综上所述，在本项目的资助下，我们开发了CRIC-seq新技术深入阐明了PTBP1二聚化介导RNA空间结构调控可变剪接的位置效应，这为探索特定RNA结合蛋白介导的RNA-RNA相互作用在基因表达调控和疾病发生中的作用机制提供了新的研究方法和思路。