Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation [SELEX]

RNA-binding proteins (RBPs) bind at different positions of pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These “positional rules” can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease. We developed a capture RIC-seq (CRIC-seq) method for the global mapping of RBP-associated in situ proximal RNA-RNA fragments. Two biological replicates of CRIC-seq libraries were generated for PTBP1 IP (+pCp), hnRNPA1 IP (+pCp), SRSF1 IP (+pCp), IgG IP (+pCp), PTBP1 IP (-pCp), hnRNPA1 IP (-pCp), and SRSF1 IP (-pCp) in HeLa cells. In addition, we also constructed two biological replicates of RIC-seq libraries for PTBP1 and hnRNPA1 knockdown cells to test the specificity of PTBP1- or hnRNPA1-associated RNA loops identified by CRIC-seq. To identify PTBP1 Cys23-mediated RNA loops and their influence on splicing regulation, we performed RNA-seq and CRIC-seq in the PTBP1-C23S mutant replaced (C23S+Dox) or not replaced (C23S-Dox) HeLa cells.

RNA结合蛋白（RNA-binding proteins, RBPs）可结合前体mRNA（pre-mRNA）分子的不同位置，以促进或抑制特定外显子的剪接使用。为阐明此类位置效应的作用机制，我们开发了捕获型RIC-seq（capture RIC-seq, CRIC-seq）技术，用于富集特定RBP相关的原位近端RNA-RNA片段并开展深度测序。我们在HeLa细胞中解析了hnRNPA1、SRSF1及PTBP1相关的近端RNA-RNA互作与调控机制。出乎意料的是，三维RNA图谱分析显示，单个内含子内与PTBP1结合的RNA环状结构，可通过加速不对称内含子切除优先促进可变外显子的剪接；而跨可变外显子的RNA环状结构则主要发挥剪接抑制作用。此类“位置规则”可准确预测PTBP1调控的剪接结果。我们进一步证实，癌症相关的剪接数量性状位点（splicing quantitative trait loci, sQTL）可通过降低前体mRNA上的PTBP1结合能力，破坏RNA环状结构，进而引发肿瘤中的异常剪接。本研究为探索RBP相关的近端RNA-RNA互作在基因调控与疾病中的功能提供了一种强有力的研究方法。我们开发了用于全局定位RBP相关原位近端RNA-RNA片段的捕获型RIC-seq（CRIC-seq）技术。针对HeLa细胞中的PTBP1免疫沉淀（immunoprecipitation, IP，+pCp）、hnRNPA1 IP（+pCp）、SRSF1 IP（+pCp）、IgG IP（+pCp）、PTBP1 IP（-pCp）、hnRNPA1 IP（-pCp）及SRSF1 IP（-pCp）样本，我们构建了两份生物学重复的CRIC-seq文库。此外，我们还为PTBP1与hnRNPA1敲低细胞构建了两份生物学重复的RIC-seq文库，以验证CRIC-seq鉴定得到的PTBP1或hnRNPA1相关RNA环状结构的特异性。为探究PTBP1 Cys23介导的RNA环状结构及其对剪接调控的影响，我们在被PTBP1-C23S突变体替换（C23S+Dox）或未被替换（C23S-Dox）的HeLa细胞中开展了RNA测序（RNA-seq）与CRIC-seq实验。