The grant funded a transformative research program that deepened understanding of how intrinsically disordered regions (IDRs) in RNA-binding proteins (RBPs) regulate RNA metabolism and contribute to neurodegenerative diseases like ALS and FTD. Mutations in IDRs often alter phase separation and ribonucleoprotein (RNP) assembly, but traditional methods like in vitro droplet assays and cellular imaging failed to explain how these changes affect RNA binding specificity in vivo. The program tackled this by pursuing five objectives: elucidating RBP functions in stem-cell and neuronal differentiation; exploring multivalent RNA features in RNP condensation; engineering RNA multivalency to manipulate assembly; perturbing IDR-dependent dynamics; and examining species-specific RNP modes.
Key technological breakthroughs included iiCLIP, an enhanced, non-radioactive iCLIP protocol for quantitative RBP-RNA interaction mapping (Lee et al., bioRxiv 2021), and the PEKA computational pipeline for motif discovery and binding region assignment (Kuret et al., Genome Biol, 2022). These tools enabled detailed analyses of RBP mutants across datasets.
Major discoveries emerged from studies on TDP-43, revealing IDR-dependent condensation as crucial for binding long, multivalent RNA regions with dispersed motifs; reduced condensation in mutants caused selective mis-splicing of 3’UTRs and autoregulatory failures (Hallegger et al., Cell, 2021). Collaborative work on UNC13A showed how variants exploit multivalent RNA to evade TDP-43 repression of cryptic exons (Brown et al., Nature, 2022). Further, mapping RBP dynamics during differentiation demonstrated how phosphorylation alters IDR assembly, rewiring mRNA binding and decay (Modic et al., NSMB, 2024).
These findings established multivalent RNA regions as scaffolds gated by IDR dynamics, culminating in the 2025 Nature discovery of "interstasis"—an intermediate state between dispersed binding and full condensation, where RBPs selectively stabilize extended RNA regions without visible aggregates. Interstasis explains how subtle IDR mutations trigger early RNA misregulation in ALS/FTD, shifting paradigms from binary models to finely tuned intermediates vital for splicing, stability, and translation.
Evolutionary analyses of codon biases in multivalent regions illuminated species-specific RNP evolution. Overall, the program’s insights and tools pave the way for therapies targeting IDR-multivalency interactions to correct pathogenic RNA processing, offering hope for selective disease intervention.
