Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins.

Chih-Yung S Lee,Andrea Putnam,Tu Lu,Shuaixin He,John Paul T Ouyang,Geraldine Seydoux

doi:10.7554/elife.52896

Abstract

RNA granules are protein/RNA condensates. How specific mRNAs are recruited to cytoplasmic RNA granules is not known. Here, we characterize the transcriptome and assembly of P granules, RNA granules in the C. elegans germ plasm. We find that P granules recruit mRNAs by condensation with the disordered protein MEG-3. MEG-3 traps mRNAs into non-dynamic condensates in vitro and binds to ~500 mRNAs in vivo in a sequence-independent manner that favors embryonic mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. Localization to P granules is not required for translational repression but is required to enrich mRNAs in the germ lineage for robust germline development. Our observations reveal similarities between P granules and stress granules and identify intrinsically-disordered proteins as drivers of RNA condensation during P granule assembly.

Highlights

RNA granules are RNA/protein condensates that assemble in the absence of limiting membranes
Phase separation of ‘naked RNA’ has been proposed to drive the assembly of stress granules, RNA granules that form under conditions of translational stress when thousands of mRNA molecules are released from polysomes (Bounedjah et al, 2014; Van Treeck and Parker, 2018)
To identify RNAs that associate with P granules in vivo, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation experiments (Huppertz et al, 2014) on MEG-3 and PGL-1 proteins tagged at each locus with GFP

Summary

Introduction

RNA granules are RNA/protein condensates that assemble in the absence of limiting membranes. Intrinsically-disordered domains in RNA-binding proteins mediate labile, multivalent protein-protein interactions that drive phase separation in vitro (Kato et al, 2012; Banani et al, 2017; Shin and Brangwynne, 2017; Boeynaems et al, 2018; Mittag and Parker, 2018). Disordered domains in proteins interact with RNA and readily phase separate with RNA in vitro (Zagrovic et al, 2018; Hentze et al, 2018), but whether these domains participate directly in mRNA recruitment in vivo has not yet been demonstrated. Our findings reveal similarities between P granules and stress granules and demonstrate a direct role for intrinsically disordered proteins in sequence-independent recruitment of mRNAs to RNA granules in vivo

Results

D P4 Germline

Discussion

Materials and methods

Funding Funder National Institutes of Health