m⁶A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins

Abstract

10 Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m 6 A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m 6 A-modified mRNAs are enriched in SGs, and that m 6 A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of 15 m 6 A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m 6 A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and 20 critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m 6 A-binding YTHDF proteins in regulating phase separation.

Binding to m6A RNA promotes YTHDF2-mediated phase separation

“…While this manuscript was in preparation, several groups reported that YTHDF family proteins display phase separation potential (Fu and Zhuang, 2019;Gao et al, 2019;Ries et al, 2019) stimulated by m 6 A RNA (Gao et al, 2019;Ries et al, 2019), consistent with our finding that m 6 A promotes the phase separation potential of YTHDF2.…”

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…These proteins may, therefore, differ in requirements for their IDRs for RNA interactions compared with other YTH proteins, or they may simply bind with higher affinity. It is an interesting property of both mammalian YTHDFs and plant ECTs that they are able to undergo phase transition in vitro to a condensed liquid or gel-like phase (Arribas-Hernández et al, 2018;Fu and Zhuang, 2019;Gao et al, 2019;Ries et al, 2019). It is of considerable interest to determine whether such phase separation properties underlie biological functions and the subcellular localization of YTHDF proteins.…”

rec-Y2H matrix screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

“…Interestingly, newly identified interactions connect the m6A-reading proteins in YTHDF1 and 3 to core components of stress granules as QKI and RBFOX2. This is of high interest as recently it was found that m6A-modified mRNAs are enriched in stress granules and that YTHDF1 and 3 are essential for their formation (Fu and Zhuang, 2019). Regarding factors involved in rRNA processing ( Figure S5E), we detect new interactions between exosome components (EXOSC7&8) and ribosomal subunits (RPL0,3,21 and RPS28) which indicate that direct interactions between ribosomal proteins and these exosome components could play a role in rRNA processing.…”