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

Area of Science:

• Cellular Biology
• Molecular Signaling
• Genomics

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses to external stimuli via second messengers like cyclic AMP (cAMP).
• Understanding GPCR/cAMP pathway regulation is crucial for deciphering cell survival, proliferation, and differentiation.
• Systematic identification of GPCR/cAMP signaling regulators has been limited.

Purpose of the Study:

• To develop and apply a high-throughput screening platform for unbiased identification of GPCR/cAMP pathway regulators.
• To discover novel molecular factors that modulate cAMP signaling cascades.
• To validate the functional significance of newly identified regulators.

Main Methods:

• Genome-wide CRISPR interference screening.
• A novel sortable transcriptional reporter for robust cAMP signaling readout.
• Analysis of sgRNA distribution across fractions to identify regulators with varying phenotypes.

Main Results:

• Identification of 45 strong and 50 moderate phenotype regulators of cAMP signaling previously unknown.
• Validation of seven novel mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, HRAS) impacting distinct pathway steps.
• Demonstration of the screening platform's efficacy in unbiased, high-throughput identification of signaling regulators.

Conclusions:

• The developed screening platform successfully identifies bona fide regulators of GPCR/second messenger cascades.
• The study highlights the extensive functional diversity among GPCR regulators.
• This work provides a foundation for further exploration of complex cellular signaling networks.