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

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. Overall design: 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）可结合至前体mRNA（pre-mRNA）分子的不同位置，以调控特定外显子（exon）的剪接选择。为解析此类位置效应的作用机制，我们开发了捕获型RIC-seq（CRIC-seq）技术，用于富集与特定RBP结合的原位近端RNA-RNA片段以开展深度测序。我们在HeLa细胞中鉴定得到了与hnRNPA1、SRSF1及PTBP1结合的近端RNA-RNA互作及调控机制。出乎意料的是，三维RNA图谱（3D RNA map）分析显示，单个内含子（intron）中与PTBP1结合的RNA环，可通过加速不对称内含子移除过程，优先促进盒式外显子（cassette exon）的剪接；而横跨盒式外显子的RNA环则主要抑制剪接反应。此类“位置规则”可精准预测PTBP1调控的剪接结局。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结合量，破坏RNA环结构，进而引发肿瘤中的异常剪接。本研究为探究RBP结合的近端RNA-RNA互作在基因调控及疾病发生中的功能提供了强有力的技术手段。

实验整体设计：我们开发了捕获型RIC-seq（CRIC-seq）技术，用于全局定位与RBP结合的原位近端RNA-RNA片段。我们在HeLa细胞中为以下组别构建了两份生物学重复的CRIC-seq文库：PTBP1免疫沉淀（immunoprecipitation, IP）（+pCp）组、hnRNPA1免疫沉淀（+pCp）组、SRSF1免疫沉淀（+pCp）组、IgG免疫沉淀（+pCp）组、PTBP1免疫沉淀（-pCp）组、hnRNPA1免疫沉淀（-pCp）组及SRSF1免疫沉淀（-pCp）组。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，用于验证CRIC-seq鉴定得到的PTBP1或hnRNPA1结合RNA环的特异性。为鉴定PTBP1半胱氨酸23（Cys23）位点介导的RNA环及其对剪接调控的影响，我们分别在PTBP1-C23S突变体诱导表达（C23S+Dox，其中Dox为多西环素（doxycycline））及未诱导表达（C23S-Dox）的HeLa细胞中开展了RNA测序（RNA-seq）与CRIC-seq实验。