Programming of protein-RNA condensation during mammalian development

Some long noncoding RNAs (lncRNAs) have a capacity to scaffold high concentration of RNA-binding proteins (RBPs) and assemble into membraneless compartments. These are characterised by a tendency for phase separation when reconstituted in vitro, they are visible as granules under light microscope and act as liquid droplets with high mobility when studied in cells. Membraneless compartments are essential for development and play a key role in human diseases associated with liquid-liquid phase separation (LLPS). It is imperative to delineate and modulate their composition in order to understand the function of these compartments. Here, we will ask how one hallmark lncRNA scaffold, NEAT1, may act as a “seed” to assemble a ribonucleoprotein complex (RNP) with high local condensation into a membraneless compartment called paraspeckles and how this impacts the differentiation propensity of human embryonic stem cells (ESCs). It namely remains unknown how any of the lncRNA-scaffolded biomolecular compartments steer cell fate decisions, which opens the next frontier. NEAT1 promotes a phase separation of RBPs, which heavily depends on the valency of interactions formed by the intrinsically disordered regions (IDRs) of RBPs. These IDRs of paraspeckle RBPs are hotspots for neurodegeneration-causing mutations and posttranslational modifications (PTMs). We propose that PTMs of IDRs dictate the capacity of RBPs to assume multiple roles in RNA regulatory networks at different developmental stages. In this proposal, we extend the toolkit to investigate how can programming of lncRNA-dependent remodelling of RNPs drive cell-fate transitions by integrating cell engineering, iCLIP, transcriptomics, proteomics, and NMR spectroscopy along with our newly developed proximity labelling method. Our approach towards elucidating mutations in IDRs that affect cell fate transitions by altering the recruitment of the corresponding RBPs to paraspecies is a bottom-up approach: we start by observing changes in composition and PTM-dependent protein interactions and then use a computational approach to infer common principles behind these changes. This narrows down the regulatory mechanism hypotheses that will be the subject of more detailed structural studies and further investigation by programming synthetic nuclear condensates in the ESC model of differentiation. We will explore two primary hypotheses: 1. The assembly of membraneless compartments is regulated, so that it is tightly coupled to cell fate transitions in human development, and        2. Modifications in IDRs affect the function of interacting RBPs, thereby regulating membraneless compartment assembly that can contribute to cell fate transitions.