The cellular prion protein PrP C is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrP C may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrP C expression. We cultured human U251-MG glioblas-toma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrP C using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrP C . When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrP C , 9 of which were confirmed by CRISPR interference as robust regulators of PrP C biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were in-verted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrP C expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrP C levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference