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

Abstract

The cellular prion protein PrPC 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 PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. 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. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC 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 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.

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

“…The cellular prion protein PrP C is encoded by the PRNP gene and is essential for the development of prion diseases (Bueler et al, 1993). Previous microRNA and siRNA screens have uncovered a complex pattern of regulated expression of PrP C (Heinzer et al, 2021; Pease et al, 2019) However, the transcription factor(s) (TFs) controlling PrP C expression remains unclear. We measured PrP C expression in a focused arrayed activation screen with the T.gonfio sublibrary encompassing all human TFs ( Figure 6A ).…”

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

“…1C). Controls and duplicates were strategically positioned for identifying and correcting any artifactual plate gradients, dispensing errors, or hotspots (Heinzer et al, 2021; Pease et al, 2019). Such gradients can arise from problems with the dispensing and aspiration steps or from temperature/humidity inhomogeneities during the incubation.…”

“…Prion assemblies were disaggregated using sodium hydroxide (NaOH, pH=14, 66.6 mM) (Peretz et al, 2001), neutralized with NaH 2 PO 4 buffer (pH=4.5, 83.3 mM) to near-neutral pH (Li, 2016), and a Förster energy transfer donor-acceptor antibody pair (Allophycocyanin-POM1 (APC-POM1), Europium-POM19 (EU-POM19)) was added (Ballmer et al ., 2017; Pease et al ., 2019; Polymenidou et al, 2008). PrP was then detected by time resolved (TR) FRET (Ballmer et al, 2017; Heinzer et al, 2021; Pease et al, 2019). We termed the resulting assay QUantItative Prion PhospholipasE pRotection assay (QUIPPER, Fig.…”

“…Instead, cells were lysed in 18 μL of lysis buffer. Finally, for all the approaches, 9 μL of TR-FRET antibody pairs, POM1 (Polymenidou et al, 2008) coupled to Allophycocyanin (APC) and POM19 coupled to Europium (EU) as previously described (Ballmer et al, 2017; Heinzer et al, 2021) were added to a final concentration of 5 and 2.5 nM, respectively. After each dispensing step during sample preparation and TR-FRET, plates were centrifuged at 1000 rpm for one minute.…”

An arrayed genome‐wide perturbation screen identifies the ribonucleoprotein Hnrnpk as rate‐limiting for prion propagation

“…In the past, such screens have been most effectively performed in unicellular organisms that undergo a haploid phase, such as yeast (Derkatch et al , 2001 ; Kryndushkin & Wickner, 2007 ). However, more recent technologies such as RNA interference (RNAi) and CRISPR have enabled the deployment of forward genetic screens in diploid mammalian cells (Mohr et al , 2010 ; Kampmann, 2018 ; Heinzer et al , 2021 ).…”