A single protein, RBPMS, acts as a master switch that rewires how smooth muscle cells stitch together their genetic instructions, and researchers want to know exactly how it does this. Every human gene can be cut and spliced into multiple different versions—a process called alternative splicing—allowing one gene to produce many distinct proteins. This splicing must be carefully controlled in different cell types, or the wrong proteins get made. RBPMS is a rare protein that turns on a specific splicing programme in smooth muscle cells, which line blood vessels and internal organs. The protein has a floppy, unstructured region that helps it clump together into tiny droplets inside cells. When these clumps go wrong, they can turn solid and contribute to disease. Current experiments mostly study this process in living cells, which makes it hard to isolate the physical rules at work. This project will use purified components in a test tube—something rarely achieved—to decode the precise molecular grammar that governs how RBPMS assembles, interacts with other proteins, and controls splicing on individual RNA molecules. This is fundamental science. A deep understanding of how RNA-binding proteins organise themselves to regulate splicing could, in the long term, inform the design of RNA-based therapeutics or reveal why mutations in these proteins lead to solid aggregates in disease.
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Alternative splicing (AS) is an important post-transcriptional process that allows individual genes to generate multiple mRNA variants encoding functionally distinct protein isoforms. Cell-type specific regulation of AS is principally achieved by the action of RNA Binding Proteins (RBPs). By binding to specific control sequences in pre-mRNA, these RBPs can either promote or inhibit assembly of splicing complexes at regulated splice sites. Many of the RBPs that regulate AS are widely expressed in different cell types, but a smaller number show more restricted expression and can act as “master regulators” that switch on specific AS programmes. Regulatory RBPs usually contain one or more structured RNA binding domains (RBDs) that allow them to recognise and bind to specific sequences in RNA. Their activity often requires additional regions without well defined structure – so called Intrinsically Disordered Regions (IDRs). In recent years it has become apparent that many of these RBPs are able to assemble into large structures that can be directly viewed by microscopy in cells. The assembly of these “biomolecular condensates” is driven by interactions between their IDRs and by multiple interactions between the RBPs and RNA. The importance of properly controlled assembly of biomolecular condensates is illustrated by the diseases that can arise from mutations in RBPs, leading to the normally liquid condensates becoming solid aggregates. The principles underlying regulation of AS by RBPs have been investigated by a number of experimental approaches, mostly using live cells (in vivo). A rigorous understanding of the physical principles underpinning the action of RBPs in regulating AS would ideally use cell-free (in vitro) approaches, but reconstituting cell-specific AS in vitro has rarely been achieved. This collaborative interdisciplinary project builds upon our recent discovery of the RBP RBPMS as a master regulator of AS in smooth muscle cells (SMCs), and the demonstration that it can confer SMC-regulation upon AS in vitro, acting as a specific repressor or activator of different target exons. RBPMS has an RBD that recognises CAC motifs in RNA and an IDR that is essential for function and confers the ability to form assemblies at different length scales, from >20 subunits (at endogenous concentrations) to microscopically visible condensates (at higher concentrations), in vitro. Our project will harness an array of state of the art approaches (a number of which were developed by the BBSRC SpliceSelect sLoLa) to address the following questions: What is the “molecular grammar” — the amino acid composition determinants — that enables splicing regulation by the IDR of RBPMS? How does the IDR mediate and control self-assembly at different length scales, and on individual RNA substrates? How does the IDR mediate interactions with other RBPs and splicing factors? How do IDR-mediated interactions orchestrate regulated splicing on individual pre-mRNA molecules? The outcome of our research will be a deep understanding of how the types of interactions that enable RBPs to assemble condensates can, in a more restrained way, allow the assembly of much smaller defined regulatory complexes on individual RNA molecules to direct regulated patterns of AS. Given the central role of RNA in normal cell function and the opportunities for development of RNA-based therapeutics, our project aligns with the Understanding the Rules of Life, Transformative Technologies and Bioscience for Health themes in BBSRC’s 2022-25 Strategic Delivery Plan.
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