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

Publication ,  Journal Article
Semesta, KM; Tian, R; Kampmann, M; von Zastrow, M; Tsvetanova, NG
Published in: PLoS Genet
October 2020

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.

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Published In

PLoS Genet

DOI

EISSN

1553-7404

Publication Date

October 2020

Volume

16

Issue

10

Start / End Page

e1009103

Location

United States

Related Subject Headings

  • Tumor Suppressor Protein p53
  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • RNA Splicing Factors
  • Proto-Oncogene Proteins p21(ras)
  • Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases
  • Nuclear Pore Complex Proteins
  • Membrane Proteins
  • Humans
 

Citation

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Semesta, K. M., Tian, R., Kampmann, M., von Zastrow, M., & Tsvetanova, N. G. (2020). A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling. PLoS Genet, 16(10), e1009103. https://doi.org/10.1371/journal.pgen.1009103
Semesta, Khairunnisa Mentari, Ruilin Tian, Martin Kampmann, Mark von Zastrow, and Nikoleta G. Tsvetanova. “A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.PLoS Genet 16, no. 10 (October 2020): e1009103. https://doi.org/10.1371/journal.pgen.1009103.
Semesta KM, Tian R, Kampmann M, von Zastrow M, Tsvetanova NG. A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling. PLoS Genet. 2020 Oct;16(10):e1009103.
Semesta, Khairunnisa Mentari, et al. “A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.PLoS Genet, vol. 16, no. 10, Oct. 2020, p. e1009103. Pubmed, doi:10.1371/journal.pgen.1009103.
Semesta KM, Tian R, Kampmann M, von Zastrow M, Tsvetanova NG. A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling. PLoS Genet. 2020 Oct;16(10):e1009103.

Published In

PLoS Genet

DOI

EISSN

1553-7404

Publication Date

October 2020

Volume

16

Issue

10

Start / End Page

e1009103

Location

United States

Related Subject Headings

  • Tumor Suppressor Protein p53
  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • RNA Splicing Factors
  • Proto-Oncogene Proteins p21(ras)
  • Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases
  • Nuclear Pore Complex Proteins
  • Membrane Proteins
  • Humans