RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3's access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.
- Tu Lu
- Geraldine Seydoux
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Julie Ahringer, University of Cambridge, United Kingdom
© 2016, Smith et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.
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