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

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 w...

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Autores principales:	Daniel Heinzer, Merve Avar, Daniel Patrick Pease, Ashutosh Dhingra, Jiang-An Yin, Elke Schaper, Berre Doğançay, Marc Emmenegger, Anna Spinelli, Kevin Maggi, Andra Chincisan, Simon Mead, Simone Hornemann, Peter Heutink, Adriano Aguzzi
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/9152f0d3e0c7477f98a6de46bbe46854
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spelling	oai:doaj.org-article:9152f0d3e0c7477f98a6de46bbe468542021-12-02T19:59:58ZNovel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.1553-73661553-737410.1371/journal.ppat.1010013https://doaj.org/article/9152f0d3e0c7477f98a6de46bbe468542021-10-01T00:00:00Zhttps://doi.org/10.1371/journal.ppat.1010013https://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374The 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.Daniel HeinzerMerve AvarDaniel Patrick PeaseAshutosh DhingraJiang-An YinElke SchaperBerre DoğançayMarc EmmeneggerAnna SpinelliKevin MaggiAndra ChincisanSimon MeadSimone HornemannPeter HeutinkAdriano AguzziPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 17, Iss 10, p e1010013 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Daniel Heinzer Merve Avar Daniel Patrick Pease Ashutosh Dhingra Jiang-An Yin Elke Schaper Berre Doğançay Marc Emmenegger Anna Spinelli Kevin Maggi Andra Chincisan Simon Mead Simone Hornemann Peter Heutink Adriano Aguzzi Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.
description	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.
format	article
author	Daniel Heinzer Merve Avar Daniel Patrick Pease Ashutosh Dhingra Jiang-An Yin Elke Schaper Berre Doğançay Marc Emmenegger Anna Spinelli Kevin Maggi Andra Chincisan Simon Mead Simone Hornemann Peter Heutink Adriano Aguzzi
author_facet	Daniel Heinzer Merve Avar Daniel Patrick Pease Ashutosh Dhingra Jiang-An Yin Elke Schaper Berre Doğançay Marc Emmenegger Anna Spinelli Kevin Maggi Andra Chincisan Simon Mead Simone Hornemann Peter Heutink Adriano Aguzzi
author_sort	Daniel Heinzer
title	Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.
title_short	Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.
title_full	Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.
title_fullStr	Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.
title_full_unstemmed	Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.
title_sort	novel regulators of prpc biosynthesis revealed by genome-wide rna interference.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/9152f0d3e0c7477f98a6de46bbe46854
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Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference.

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