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Splicing Kinetics and Coordination Revealed by Direct Nascent RNA Sequencing through Nanopores.
Molecular Cell ( IF 14.5 ) Pub Date : 2019-12-12 , DOI: 10.1016/j.molcel.2019.11.017
Heather L Drexler 1 , Karine Choquet 1 , L Stirling Churchman 1
Affiliation  

Understanding how splicing events are coordinated across numerous introns in metazoan RNA transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of co-transcriptional processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. Long nano-COP reads reveal that, in human and Drosophila cells, splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B rapidly diminished global co-transcriptional splicing. We found that splicing order does not strictly follow the order of transcription and is associated with cis-acting elements, alternative splicing, and RNA-binding factors. Further, neighboring introns in human cells tend to be spliced concurrently, implying that splicing of these introns occurs cooperatively. Thus, nano-COP unveils the organizational complexity of RNA processing.

中文翻译:


通过纳米孔直接新生 RNA 测序揭示剪接动力学和协调。



了解后生动物 RNA 转录本中众多内含子之间的剪接事件如何协调,需要对活细胞中的瞬时 RNA 加工事件进行定量分析。我们开发了共转录处理的纳米孔分析 (nano-COP),其中新生 RNA 通过纳米孔直接测序,揭示了 RNA 剪接的动态和模式,而没有扩增引入的偏差。长纳米 COP 读数显示,在人类和果蝇细胞中,剪接发生在 RNA 聚合酶 II 转录数千碱基的前 mRNA 后,表明后生动物剪接发生在转录机器的远端。分支位点识别复合物 SF3B 的抑制迅速减少了全局共转录剪接。我们发现剪接顺序并不严格遵循转录顺序,而是与顺式作用元件、选择性剪接和RNA结合因子相关。此外,人类细胞中相邻的内含子往往同时剪接,这意味着这些内含子的剪接是协同发生的。因此,nano-COP 揭示了 RNA 加工的组织复杂性。
更新日期:2019-12-13
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