Highlights d Direct RNA sequencing exposes splicing dynamics without biases from amplification d Splicing occurs after RNA Pol II transcribes kilobases past introns in metazoans d Introns are frequently removed in a defined order that does not follow transcription d Splicing in humans is coordinated across proximal introns

No abstract

Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes' transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.

Human genes have numerous exons that are differentially spliced within pre-mRNA.Understanding how multiple splicing events are coordinated across nascent 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. nano-COP showed that in both human and Drosophila cells, co-transcriptional 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 globally abolished co-transcriptional splicing in both species. Our findings revealed that splicing order does not strictly follow the order of transcription and is influenced by cis-regulatory elements. In human cells, introns with delayed splicing frequently neighbor alternative exons and are associated with RNA-binding factors. Moreover, neighboring introns in human cells tend to be spliced concurrently, implying that splicing occurs cooperatively. Thus, nano-COP unveils the organizational complexity of metazoan RNA processing.