Novel regulators of PrP^Cbiosynthesis revealed by genome-wide RNA interference

Abstract

The availability of the cellular prion protein PrPC is limiting to prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the proteins and the biochemical pathways controlling the biosynthesis and the degradation of PrPC may represent therapeutic targets. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC. We cultured human U251-MG glioblastoma cells in the presence of 64’752 unique siRNAs targeting 21’584 annotated human genes, and measured PrPC using a one-pot fluorescence resonance energy transfer immunoassay in 51’128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC, which were then filtered through multiple secondary screens. Recursive candidate attrition yielded 54 novel regulators of PrPC, nine of which emerged as robust regulators of PrPC biosynthesis and degradation by transcriptional suppression in a CRISPR-interference validation screen. Six candidates were found to regulate PrPC in the opposite direction when transcriptionally activated using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent modulator of PrPC through the degradation of PRNP mRNA. Because of its hypothesis-free design, the present listing paints an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.