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


ABSTRACT: 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.

SUBMITTER: Drexler HL 

PROVIDER: S-EPMC7060811 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Drexler Heather L HL   Choquet Karine K   Churchman L Stirling LS  

Molecular cell 20191212 5


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  ...[more]

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