Structural and phase-separation investigations of the hnRNPs and viral RNPs

RNA is a very versatile molecule as it is in essence single stranded having the possibility to fold into itself or bind proteins. While DNA compaction is fairly well understood and structurally characterized at atomic level, RNA compaction is much less understood possibly due to the dynamic nature of RNA as it can exchange between multiple conformation and be bound by thousands RNA binding proteins (RBPs). Yet, RNA does compact as seen with viral RNA encapsulated in viral capsid or as part of pre-mRNA particle like the 40S ribonucleosomes. Studying RNA packaging faces an other obstacle in the fact that RNA alone and RNA-protein complexes tend to phase-separate at physiological conditions (concentration and buffer conditions), rendering studies of RNA compaction by RNA binding proteins very difficult to do. We postulate here that RNA compaction by RBPs requires a phase-separation step in order to concentrate and compact the RNA. This phase-separation step is then followed by a maturation step allowing the ribonucleoparticles to reach a stable conformation. As model systems, we are studying the 40S ribonucleosome which is a particle postulated to compact introns in human cells and the SARS-CoV2 Nucleocapsid (Nu) which is postulated to compact the viral RNA genome into its capsid during virion formation. We recently rediscovered the existence of the 40S ribonucleosome which has been postulated in the 60-70s to be the compacting unit of pre-mRNA. We also found out that region of the proteins important for ribonucleosome formation are also crucial for liquid-liquid phase separation (LLPS). We therefore aim at understanding the LLPS properties of the major components of the ribonucleosome (namely the protein hnRNPC1/2, hnRNPA1/2, hnRNPB1/2 with and without RNA), how the droplet mature over time and ultimately how they fold into a 40S compacted particle. Similarly, we aim at understanding the LLPS properties of the SARS-Cov2 Nu that phase separates in the presence of RNA but also of cognate proteins and nucleotides. Nu phase separation is critical for the viral gene expression and its encapsidation. We therefore will study droplet formation of Nu with RNA and various binding partners, follow the maturation of the droplets and observe if the droplet can mature into folded ribonucleoparticles.