Co-transcriptional splicing efficiencies differ within genes and between cell types

Pre-mRNA splicing is carried out by the spliceosome and involves splice site recognition, removal of introns, and ligation of exons. Components of the spliceosome have been shown to interact with the elongating RNA polymerase II (RNAPII), which is thought to allow splicing to occur concurrently with transcription. However, little is known about the regulation and efficiency of cotranscriptional splicing in human cells. In this study, we used Bru-seq and BruChase-seq to determine the cotranscriptional splicing efficiencies of 17,000 introns expressed across six human cell lines. We found that less than half of all introns across these six cell lines were cotranscriptionally spliced. Splicing efficiencies for individual introns showed variations across cell lines, suggesting that splicing may be regulated in a cell type–specific manner. Moreover, the splicing efficiency of introns varied within genes. The efficiency of cotranscriptional splicing did not correlate with gene length, intron position, splice site strengths, or the intron/neighboring exons GC content. However, we identified binding signals from multiple RNA binding proteins (RBPs) that correlated with splicing efficiency, including core spliceosomal machinery components—such as SF3B4, U2AF1, and U2AF2 showing higher binding signals in poorly spliced introns. In addition, multiple RBPs, such as BUD13, PUM1, and SND1, showed preferential binding in exons that flank introns with high splicing efficiencies. The nascent RNA splicing patterns presented here across multiple cell types add to our understanding of the complexity in RNA splicing, wherein RNA-binding proteins may play important roles in determining splicing outcomes in a cell type– and intron-specific manner. Overall design: Nascent RNA Bru-seq and BruChase-seq used to determine co-transcriptional splicing efficiencies of 17000 introns expressed across 6 diverse human cell lines

前信使RNA（pre-mRNA）剪接由剪接体（spliceosome）介导完成，该过程涵盖剪接位点识别、内含子切除与外显子连接。已有研究证实，剪接体组分可与延伸中的RNA聚合酶II（RNAPII）发生相互作用，这一机制被认为可实现剪接与转录的同步进行。然而，目前学界对人类细胞中共转录剪接的调控机制与效率仍缺乏深入认知。本研究采用Bru-seq与BruChase-seq技术，对六种人类细胞系中表达的17000个内含子的共转录剪接效率开展了系统性测定。研究结果显示，上述六种细胞系中仅不足半数的内含子可通过共转录途径完成剪接。单个内含子的剪接效率在不同细胞系间存在显著差异，这提示剪接过程可能以细胞类型特异性的方式受到调控。此外，同一基因内不同内含子的剪接效率也存在明显异质性。共转录剪接效率与基因长度、内含子位置、剪接位点强度以及内含子/邻近外显子的GC含量均无显著相关性。但本研究鉴定出多个与剪接效率密切相关的RNA结合蛋白（RNA binding protein, RBP）结合信号：核心剪接体组分如SF3B4、U2AF1及U2AF2在剪接效率较低的内含子上的结合信号更强；而BUD13、PUM1与SND1等多种RNA结合蛋白则优先结合于剪接效率较高的内含子两侧的外显子区域。本研究提供的多细胞类型新生RNA剪接特征，进一步加深了学界对RNA剪接复杂性的理解，其中RNA结合蛋白可能以细胞类型与内含子特异性的方式在决定剪接结局中发挥关键作用。

整体实验设计：本研究通过新生RNA Bru-seq与BruChase-seq技术，对六种不同人类细胞系中表达的17000个内含子的共转录剪接效率进行了测定。