A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights in...

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Autores principales:	Khairunnisa Mentari Semesta, Ruilin Tian, Martin Kampmann, Mark von Zastrow, Nikoleta G Tsvetanova
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2020
Materias:	Genetics QH426-470
Acceso en línea:	https://doaj.org/article/630edfde867848fbacb1414356ff24bb
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spelling	oai:doaj.org-article:630edfde867848fbacb1414356ff24bb2021-12-02T20:02:47ZA high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.1553-73901553-740410.1371/journal.pgen.1009103https://doaj.org/article/630edfde867848fbacb1414356ff24bb2020-10-01T00:00:00Zhttps://doi.org/10.1371/journal.pgen.1009103https://doaj.org/toc/1553-7390https://doaj.org/toc/1553-7404G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.Khairunnisa Mentari SemestaRuilin TianMartin KampmannMark von ZastrowNikoleta G TsvetanovaPublic Library of Science (PLoS)articleGeneticsQH426-470ENPLoS Genetics, Vol 16, Iss 10, p e1009103 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Genetics QH426-470
spellingShingle	Genetics QH426-470 Khairunnisa Mentari Semesta Ruilin Tian Martin Kampmann Mark von Zastrow Nikoleta G Tsvetanova A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.
description	G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.
format	article
author	Khairunnisa Mentari Semesta Ruilin Tian Martin Kampmann Mark von Zastrow Nikoleta G Tsvetanova
author_facet	Khairunnisa Mentari Semesta Ruilin Tian Martin Kampmann Mark von Zastrow Nikoleta G Tsvetanova
author_sort	Khairunnisa Mentari Semesta
title	A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.
title_short	A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.
title_full	A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.
title_fullStr	A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.
title_full_unstemmed	A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.
title_sort	high-throughput crispr interference screen for dissecting functional regulators of gpcr/camp signaling.
publisher	Public Library of Science (PLoS)
publishDate	2020
url	https://doaj.org/article/630edfde867848fbacb1414356ff24bb
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A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.

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