6. What controls the timing of splicing?

How do cells decide when a newly synthesised RNA molecule is ready to be spliced? Before messenger RNA (mRNA) can be translated into protein, non-coding regions called introns must be removed through a process known as RNA splicing. It has long been thought that much of this processing occurs while RNA polymerase II is still transcribing the gene, allowing introns to be removed shortly after they are synthesised.

Our recent research challenges this conventional view. Using long-read nanopore sequencing to examine newly synthesised RNA associated with RNA polymerase II in human cells and mouse brain, we discovered that splicing is often substantially delayed during transcription. Many introns remain unspliced even after RNA polymerase II has travelled thousands of nucleotides beyond them, while extensive splicing becomes apparent in RNA molecules that have undergone 3′-end processing and polyadenylation (Sethi et al., 2025, bioRxiv; doi:10.1101/2025.04.27.650906).

These findings raise a fundamental question: what determines when the spliceosome, the molecular machinery responsible for removing introns, becomes catalytically active? Does splicing simply proceed slowly, or do cells actively delay its completion until a particular stage of RNA maturation? One intriguing possibility is that interactions between RNA polymerase II, the spliceosome and other RNA-processing factors act as a molecular checkpoint to coordinate transcription and splicing.

In this project, we aim to uncover the molecular mechanisms that control the timing of splicing catalysis. Building on our established methods for isolating RNA polymerase II and its associated RNA and proteins, we will use biochemical purification and mass spectrometry to identify factors that connect transcription with the splicing machinery. We will then investigate how candidate proteins influence spliceosome activity and the timing of intron removal, combining genetic perturbations with long-read sequencing of newly synthesised RNA.

Ultimately, we hope to reveal how cells coordinate transcription and RNA processing, and whether the timing of splicing represents an important, previously underappreciated layer of gene expression regulation.

Techniques: Mammalian cell culture, CRISPR-based genome engineering, protein immunoprecipitation and biochemical purification, mass spectrometry, nanopore direct RNA sequencing, and computational analysis of RNA processing.