In eukaryotes, genes are transcribed by RNA polymerase-II (Pol-II) and introns are removed by the spliceosome largely cotranscriptionally^1,2,3; analysis using long-read sequencing revealed that splicing occurs immediately after Pol-II passes introns in yeast^4,5. Here, we developed a Nanopore-based method to profile chromatin-bound RNA that enables the simultaneous detection of splicing status, Pol-II position and polyadenylation at the genome-wide scale in Arabidopsis. We found that more than half of the introns remain unspliced after Pol-II transcribes 1 kb past the 3′ splice site, which is much slower than the rate of splicing reported in yeast^4,5. Many of the full-length chromatin-bound RNA molecules are polyadenylated, yet still contain unspliced introns at specific positions. These introns are nearly absent in the cytoplasm and are resistant to nonsense-mediated decay, suggesting that they are post-transcriptionally spliced before the transcripts are released into the cytoplasm; we therefore termed these introns post-transcriptionally spliced introns (pts introns). Analysis of around 6,500 public RNA-sequencing libraries found that the splicing of pts introns requires the function of splicing-related proteins such as PRMT5 and SKIP, and is also influenced by various environmental signals. The majority of the intron retention events in Arabidopsis are at pts introns, suggesting that chromatin-tethered post-transcriptional splicing is a major contributor to the widespread intron retention that is observed in plants, and could be a mechanism to produce fully spliced functional mRNAs for rapid response.

在真核生物中，基因被RNA聚合酶II（Pol-II）转录，内含子被剪接体在很大程度上共转录^1,2,3去除；使用长时间阅读测序的分析显示，Pol-II通过酵母^4,5中的内含子后立即发生剪接。在这里，我们开发了一种基于纳米孔的方法来分析染色质结合的RNA，该方法能够在拟南芥全基因组范围内同时检测剪接状态，Pol-II位置和聚腺苷​​酸化。我们发现，在Pol-II转录超过3'剪接位点1 kb之后，超过一半的内含子仍未剪接，这比酵母^4,5中报道的剪接速度慢得多。许多全长染色质结合的RNA分子是聚腺苷酸化的，但在特定位置仍包含未剪接的内含子。这些内含子几乎不存在于细胞质中，并且对无义介导的衰变具有抗性，表明它们在转录本释放到细胞质中之前被转录后剪接。因此，我们将这些内含子称为转录后剪接的内含子（pts内含子）。对大约6,500个公共RNA测序文库的分析发现，pts内含子的剪接需要剪接相关蛋白（如PRMT5和SKIP）的功能，并且还受到各种环境信号的影响。拟南芥中大多数内含子保留事件 是在pts内含子处，提示染色质栓系的转录后剪接是植物中观察到的广泛内含子保留的主要因素，并且可能是产生完全剪接的功能性mRNA以快速响应的机制。