Direct sequencing of nascent RNA exposes splicing kinetics and order | Heather Drexler
London Calling 2019
Heather Drexler (Harvard Medical School) began her talk by highlighting the complexity of the process of RNA splicing. Using the human Dystrophin gene as an example, she demonstrated how human genes contain long introns with degenerate sequences at the splice sites; these must be spliced out and the distant exons joined, producing the correct alternative mRNA isoform for each cell type. She also noted that disrupted mechanisms in this process can lead to disease, such as muscular dystrophy in the example of the Dystrophin gene. The processes of transcription and splicing are "physically and mechanistically coupled", and splicing happens ~10x faster in vivo, interacting with transcription machinery within cells. Splicing factors interact with RNA polymerase II (Pol II), and mutations in the latter cause splicing defects. Pol II elongation rate, therefore, regulates alternative splicing. Heather displayed an electron micrograph showing loops of RNA forming, demonstrating the process of splicing. She asked: how is this occurring in living cells?
Whilst most studies looking at the relationship between transcription and splicing focus on the time between these processes, Heather and her team decided to investigate the distance between them. Firstly, they developed a method of enriching for unspliced nascent RNAs, via a strategy combining cellular fractionation and metabolic labelling; Heather described how combining the two strategies brought the benefits of both. The nascent RNA was then polyA-tailed via E. coli polyA polymerase, then the native RNA prepared for sequencing using Oxford Nanopore's Direct RNA sequencing kit; Heather noted that "a great benefit of this is that I don't have to rely on PCR", preventing size biases that would distort splicing measurements. The team named the method nano-COP: nanopore analysis of CO-transcriptional Processing. The method exposed the nascent transcriptome, producing sequences for RNA that was still in the process of being spliced; Heather showed the wide range of transcripts produced, their different structures illuminating the order in which splicing occurs. She also showed that the length of the polyA tail could be determined from the native RNA reads; a difference in tail length was observed between artificially added and endogenous polyA tails.
Heather then asked: "how far does Pol II transcribe before splicing?". The data indicated that, in humans, very little splicing occurred when Pol II was within 2 kb. However, in Drosophila, transcription occurred much closer to splicing; Heather explained that Drosophila has a very different gene structure, with shorter introns, which could explain the more rapid splicing. Next, the team added the splicing inhibitor Pladienolide B, to interrupt splicing. The resulting data showed that both human and Drosophila samples treated with PlaB showed very little splicing, whilst splicing was observed in those without the splicing inhibitor, validating nano-COP's detection of early transcriptional dynamics. They then investigated whether splicing order follows the direction of transcription. In a comparison of whether upstream or downstream introns are more likely to be spliced, human splicing was relatively even between the two, whilst in Drosophila, upstream introns are more likely to be spliced. In Drosophila, introns neighbouring each other were also seen to have more similar splicing dynamics. In both humans and Drosophila, constitutive exons were shown to have more rapid splicing, with more of a delay for alternate exons.
Heather summarised by saying that the nano-COP project enabled the direct sequencing of nascent RNAs, allowing the observation of patterns of early RNA processing. In human cells, Pol II was shown to transcribe for several kilobases prior to intron splicing; the process of splicing was seen to not always follow the order of transcription and is coordinated. Finally, introns that neighbour alternative exons were seen to have delayed splicing kinetics. Next up, the team plan to optimise nano-COP for longer reads and greater coverage to investigate longer-range interactions. They also intend to study how perturbation of transcription directly affects splicing.
Check us out:
Website - https://nanoporetech.com/
Twitter - / nanopore
Facebook - / oxford-nanopore-technologies-251034380246
LinkedIn - / oxford-nanopore-technologies
Instagram - / oxfordnanopore
