Department of Biological Sciences, California State University Fullerton, Fullerton, California 92831, United States.
Department of Chemistry and Biochemistry, California State University Fullerton, Fullerton, California 92831, United States.
Biochemistry. 2020 Dec 22;59(50):4766-4774. doi: 10.1021/acs.biochem.0c00664. Epub 2020 Dec 7.
Polypyrimidine tract binding protein 1 (PTBP1) is a well-studied RNA binding protein that serves as an important model for understanding molecular mechanisms underlying alternative splicing regulation. PTBP1 has four RNA binding domains (RBDs) connected via linker regions. Additionally, PTBP1 has an N-terminal unstructured region that contains nuclear import and export sequences. Each RBD can bind to pyrimidine rich elements with high affinity to mediate splicing activity. Studies support a variety of models for how PTBP1 can mediate splicing regulation on target exons. Obtaining a detailed atomic view hinges on determining a crystal structure of PTBP1 bound to a target RNA transcript. Here, we created a minimal functional PTBP1 with deletions in both linker 1 and linker 2 regions and assayed for activity on certain regulated exons, including the c-Src N1 exon. We show that for a subset of PTBP1-regulated exons the linker regions are not necessary for splicing repression activity. Gel mobility shift assays reveal the linker deletion mutant binds with 12-fold higher affinity to a target RNA sequence compared to wild-type PTBP1. A minimal PTBP1 that also contains an N-terminal region deletion binds to a target RNA with an affinity higher than that of wild-type PTBP1. Moreover, this minimal protein oligomerizes readily to form a distinct higher-order complex previously shown to be required for mediating splicing repression. This minimal functional PTBP1 protein can serve as a candidate for future structure studies to understand the mechanism of splicing repression for certain regulated exons.
多嘧啶tract 结合蛋白 1（PTBP1）是一种研究充分的 RNA 结合蛋白，是理解可变剪接调控的分子机制的重要模型。PTBP1 有四个 RNA 结合结构域（RBD），通过连接区连接。此外，PTBP1 有一个包含核输入和输出序列的无规卷曲区的 N 端。每个 RBD 都可以与富含嘧啶的元件结合，亲和力很高，从而介导剪接活性。研究支持了多种关于 PTBP1 如何调节靶外显子剪接的模型。获得详细的原子视图取决于确定与靶 RNA 转录本结合的 PTBP1 的晶体结构。在这里，我们构建了一个在连接区 1 和连接区 2 都缺失的最小功能 PTBP1，并在某些受调控的外显子（包括 c-Src N1 外显子）上检测其活性。我们发现，对于一组 PTBP1 调节的外显子，连接区对于剪接抑制活性不是必需的。凝胶迁移位移分析显示，与野生型 PTBP1 相比，连接区缺失突变体与靶 RNA 序列的结合亲和力高 12 倍。还含有 N 端缺失的最小 PTBP1 与靶 RNA 的结合亲和力高于野生型 PTBP1。此外，这种最小的蛋白质很容易寡聚形成一个以前显示对介导剪接抑制所必需的独特的高级别复合物。这种最小的功能性 PTBP1 蛋白可以作为未来结构研究的候选物，以了解某些受调控的外显子的剪接抑制机制。
