Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA binding proteins with prion-like domains

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and ALS-patient derived motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders. Overall design: A common feature of ALS/FTD is the cytoplasmic mislocalization and aggregation of nuclear RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as TDP-43 or FUS, in degenerating neurons. A key therapeutic innovation for ALS/FTD would be to develop agents that reverse the aberrant cytoplasmic aggregation of TDP-43 and FUS, and return these proteins to native form and nuclear function. We aimed to identify short specific RNAs (24-48nts) that antagonize aberrant FUS assembly. While extracting recombinant GST-FUS from E. coli, we found that purified GST-FUS was bound to RNA. We sought to identify enriched RNA motifs in this FUS-bound RNA population as they might antagonize aberrant FUS assembly. RNA extraction followed by electrophoresis revealed a population of RNAs with a size range of ~50-100nts. From this RNA population, a cDNA library was constructed and sequenced. We identified 42 enriched motifs between 8-12nts in our library.