Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference
| Autor: | Simon Mead, Simone Hornemann, Marc Emmenegger, Elke Schaper, Kevin Maggi, Peter Heutink, Jiang-An Yin, Anna Spinelli, Ashutosh Dhingra, Daniel Heinzer, Berre Doğançay, Merve Avar, Daniel Patrick Pease, Adriano Aguzzi, Andra Chincisan |
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| Přispěvatelé: | University of Zurich, Bartz, Jason C, Aguzzi, Adriano |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Genetic Screens
Small interfering RNA animal diseases Gene Identification and Analysis 2405 Parasitology Biochemistry Fluorophotometry Prion Diseases Guide RNA Medical Conditions Spectrum Analysis Techniques RNA interference Zoonoses Medicine and Health Sciences Fluorescence Resonance Energy Transfer Biology (General) 2404 Microbiology RNA-Binding Proteins Phenotype Cell biology Nucleic acids Infectious Diseases Spectrophotometry RNA Interference biosynthesis [PrPC Proteins] Research Article QH301-705.5 Immunoblotting Immunology physiology [Gene Expression Regulation] 10208 Institute of Neuropathology Molecular Probe Techniques Repressor 610 Medicine & health Context (language use) Biology Transfection Research and Analysis Methods metabolism [RNA-Binding Proteins] Microbiology Cell Line 1311 Genetics Gene Types Virology mental disorders 1312 Molecular Biology Genetics Humans PrPC Proteins ddc:610 Non-coding RNA Molecular Biology Techniques Molecular Biology Gene 2403 Immunology CRISPR interference Biology and life sciences RC581-607 Gene regulation nervous system diseases Gene Expression Regulation 2406 Virology RNA Regulator Genes 570 Life sciences biology Parasitology Human genome Gene expression Immunologic diseases. Allergy Genome-Wide Association Study |
| Zdroj: | PLoS Pathogens, Vol 17, Iss 10 (2021) PLoS pathogens 17(10), e1010013 (2021). doi:10.1371/journal.ppat.1010013 PLoS Pathogens, Vol 17, Iss 10, p e1010013 (2021) PLoS Pathogens |
| ISSN: | 1553-7374 1553-7366 |
| DOI: | 10.1371/journal.ppat.1010013 |
| Popis: | 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. Author summary The cellular prion protein (PrPC) acts as both, the substrate for prion formation and mediator of prion toxicity during the progression of all prion diseases. Suppressing the levels of PrPC is a viable therapeutic strategy as PRNP null animals are resistant to prion disease and the knockout of PRNP is not associated with any severe phenotypes. Motivated by the scarcity of knowledge regarding the molecular regulators of PrPC biosynthesis and degradation, which might serve as valuable targets to control its expression, here, we present a cell-based genome wide RNAi screen in arrayed format. The screening effort led to the identification of 54 regulators, nine of which were confirmed by an independent CRISPR-based method. Among the final nine targets, we identified PUM1 as a regulator of PRNP mRNA by acting on the 3’UTR promoting its degradation. The newly identified factors involved in the life cycle of PrPC provided by our study may also represent themselves as therapeutic targets for the intervention of prion diseases. |
| Databáze: | OpenAIRE |
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