Splicing Kinetics and Coordination Revealed by Direct Nascent RNA Sequencing through Nanopores

Mol Cell. 2020 Mar 5;77(5):985-998.e8. doi: 10.1016/j.molcel.2019.11.017. Epub 2019 Dec 12.


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.

Keywords: co-transcriptional splicing; exon definition; intron definition; nano-COP; nanopore sequencing; nascent RNA; pladienolide B; splicing coordination; splicing order.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster
  • Humans
  • Introns
  • K562 Cells
  • Kinetics
  • Nanopore Sequencing*
  • Nanopores*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • RNA Precursors / genetics
  • RNA Precursors / metabolism*
  • RNA Splicing Factors / genetics
  • RNA Splicing Factors / metabolism
  • RNA Splicing*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Sequence Analysis, RNA / methods*
  • Transcription, Genetic
  • Transcriptome*


  • Drosophila Proteins
  • RNA Precursors
  • RNA Splicing Factors
  • RNA, Messenger
  • RNA Polymerase II