The role of YTHDF phase separation in the regulation of m6A mRNA stability

项目总结 N6-甲基腺苷(M6A)是转录组中含量最丰富的核苷酸修饰，已被 与发育和癌症有关。M6A的一个主要功能是降低mRNAs的稳定性。在 在细胞质中，m6A与YTHDF蛋白结合，YTHDF蛋白由两个结构域组成：一个结合YTH结构域 M6A和一个低复杂性结构域，富含促进无序的氨基酸。低复杂度域允许 YTHDF蛋白质将经历“相分离”，这是一种富含蛋白质和RNA的液滴现象 在细胞质中形成。通过与富含m6A的mRNAs形成多价相互作用，YTHDF介导相 分离允许将m6A-mRNAs隔离在细胞中相分离的液滴中。然而，它并不是 知道YTHDF蛋白的相分离潜力是如何控制的，或者这个过程是否有助于 M6A基因不稳定。对YTHDF蛋白低复杂结构域的分析揭示了几个类似Pron的结构域 结构域和可能的磷酸化位点。在这项提议中，我试图解决以下关键问题：如何 YTHDF的蛋白结构域和磷酸化是否控制相分离，并且是相 M6A mRNA降解所需的分离？为了探索这一点，我将首先确定基因突变是如何 YTHDF蛋白低复杂结构域和可能的磷酸化位点影响体外相分离 潜力。这些实验将确定某些结构域和磷酸化位点在 增强和/或降低YTHDF的相分离电势。第二，我将测试这些 突变会影响YTHDF在活细胞中的相分离和定位。这些实验将展示如何 体外相分离电位的变化影响细胞定位和相分离电位 细胞中的YTHDF。第三，我将确定YTHDF的这些突变对物种丰度的影响 以及在缺乏内源性YTHDF蛋白表达的细胞中m6A mRNAs的稳定性。这将测试是否 相分离电位和/或磷酸化位点改变的YTHDF影响m6A mRNA的衰变。 总的来说，拟议中的实验将测试是否存在影响YTHDF相分离潜力的突变 和/或细胞内定位改变m6A mRNAs的稳定性，以及这一过程是否能被YTHDF调节 磷酸化。这项研究的结果将极大地提高我们对 YTHDF蛋白和相分离对m6A基因的稳定性有影响。

PROJECT SUMMARY 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.