Abstract
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.
Highlights
ribonucleic acid (RNA) granules are concentrated assemblies of RNA and RNA-binding proteins that form without a limiting membrane in the cytoplasm or nucleoplasm of cells (Courchaine, 2016)
To determine the interdependence of these localizations, we examined the effect of removing MEX-5/6, MEG-3/4 or PGL-1/3 using RNA-mediated interference (RNAi) or genetic mutants (GLH-1 has already been shown to depend on PGL-1/3 for asymmetry [Hanazawa et al, 2011])
In zygotes derived from mothers treated with double-stranded RNA against mex-5 and mex-6 (mex-5/6(RNAi) zygotes), the MEG-3 gradient did not form and MEG-3 and PGL-1 granules remained uniformly distributed throughout the cytoplasm (Figure 1C, (Gallo et al, 2010)
Summary
RNA granules are concentrated assemblies of RNA and RNA-binding proteins that form without a limiting membrane in the cytoplasm or nucleoplasm of cells (Courchaine, 2016). Like RNA granules in vivo, proteins in LLPS droplets and hydrogels exchange with the solvent (Kato et al, 2012; Li et al, 2012; Elbaum-Garfinkle et al, 2015; Lin et al, 2015). The nucleus and many other organelles form inside layers of membrane that physically separate them from the rest of the cell Some organelles, such as the germ granule, do not have a membrane. The experiments show that a protein called MEG-3 is required to allow the components of granules to transition from behaving like individual molecules dissolved in water (similar to being dissolved in cell fluid) to assembling into droplets. Our findings are consistent with a model whereby MEX-5 antagonizes MEG-3’s access to RNA to inhibit MEG-3 condensation in the anterior cytoplasm
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