Abstract A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35’364 ternary pools of 52’746 siRNAs targeting 17’582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrP Sc , and its cellular counterpart PrP C , 40 genes affected selectively PrP Sc . Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrP C . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation