N6-methyladenosine (m6a) is the most abundant nucleotide modification in the transcriptome and has been implicated in development and cancer. A major function of m6A is to reduce the stability of mRNAs. In the cytoplasm, m6A is bound by the YTHDF proteins, which consist of two domains: a YTH domain, which binds m6A, and a low complexity domain, rich in disorder-promoting amino acids. The low complexity domain allows the YTHDF proteins to undergo `phase separation,' a phenomenon in which protein- and RNA-rich droplets form in the cytoplasm. By forming multivalent interactions with mRNAs rich in m6A, YTHDF-mediated phase separation allows the sequestration of m6A-mRNAs in phase-separated droplets in cells. However, it is not known how the phase separation potential of the YTHDF proteins is controlled or if this process contributes to m6A mRNA instability. Analysis of the low complexity domain of the YTHDF proteins reveals several prion-like domains and putative phosphorylation sites. In this proposal, I seek to address the following key question: how do the prion-like domains and phosphorylation of the YTHDFs control phase separation, and is phase separation necessary for m6A mRNA degradation? To explore this, I will first determine how mutations in the low complexity domain and at putative phosphorylation sites of YTHDF proteins affect in vitro phase separation potential. These experiments will determine the importance of certain domains and phosphorylation sites in enhancing and/or reducing the phase separation potential of the YTHDFs. Second, I will test how these mutations affect YTHDF phase separation and localization in living cells. These experiments will show how changes in in vitro phase separation potential affect cellular localization and phase separation potential of the YTHDFs in cells. Third, I will determine the effect that these mutations in the YTHDFs have on the abundance and stability of m6A mRNAs in cells lacking endogenous YTHDF protein expression. This will test if the YTHDFs with altered phase separation potential and/or phosphorylation sites influence m6A mRNA decay. Collectively, the proposed experiments will test if mutations that affect YTHDF phase separation potential and/or localization in cells alters the stability of m6A mRNAs, and if this process can be regulated by YTHDF phosphorylation. The outcomes of this study will substantially improve our understanding of the role that the YTHDF proteins and phase separation play in influencing m6A mRNA stability.
N6-methyladenosine (m6A) is a nucleotide modification in mRNA that reduces mRNA stability, and deregulation of m6A has been linked to development and cancer. The YTHDF proteins that bind to m6A can `phase separate', or form droplets in the cytoplasm, creating m6A mRNA-rich droplets in the cell. These studies will determine how phase separation modulates m6A mRNA stability and may lead to the discovery of new pathways that allow for therapeutic intervention in m6A-linked diseases.