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

YTHDF相分离在m6A mRNA稳定性调节中的作用

• 批准号: 10223884
• 负责人: Ryan J Ries
• 金额: $ 4.6万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2023-07-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223884
• 关键词: Address; Affect; Amino Acids; Binding; Binding Proteins; Biological Assay; Biology; Cell Line; Cell physiology; Cells; Cellular Stress; Cellular Structures; Cytoplasm; Data; Development; Disease; Environment; Excision; Goals; Impairment; In Vitro; Knock-out; Lead; Link; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Modification; Mutation; Nucleotides; Outcome Study; Pathway interactions; Phase; Phosphorylation; Phosphorylation Site; Play; Positioning Attribute; Process; Protein Family; Proteins; RNA; Regulation; Research Personnel; Research Proposals; Role; Series; Site; Stress; Testing; Therapeutic Intervention; Time; Training; biophysical properties; career; experimental study; improved; in vitro Assay; mRNA Decay; mRNA Instability; mRNA Stability; mRNA Transcript Degradation; mutant; next generation sequencing; prion-like; protein expression; recruit; response; skills; stem cell differentiation; stress granule; transcriptome; transcriptome sequencing

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.

项目摘要 N6-甲基腺苷（m6 a）是转录组中最丰富的核苷酸修饰， 与发育和癌症有关m6 A的主要功能是降低mRNA的稳定性。在 在细胞质中，m6 A被YTHDF蛋白结合，YTHDF蛋白由两个结构域组成： m6 A和低复杂性结构域，富含促进疾病的氨基酸。低复杂度域允许 YTHDF蛋白质经历“相分离”，这是一种富含蛋白质和RNA的液滴 在细胞质中形成。通过与富含m6 A的mRNA形成多价相互作用，YTHDF介导的阶段 分离允许将m6 A-mRNA隔离在细胞中相分离的液滴中。但不 已知如何控制YTHDF蛋白质的相分离潜力，或者该过程是否有助于 m6 A mRNA不稳定性。对YTHDF蛋白质的低复杂性结构域的分析揭示了几个朊病毒样结构域。 结构域和推定的磷酸化位点。在本建议中，我试图解决以下关键问题： 朊病毒样结构域和YTHDFs的磷酸化是否控制相分离， m6 A mRNA降解所需的分离？为了探索这一点，我将首先确定 YTHDF蛋白的低复杂性结构域和推定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的重要性， 增强和/或降低YTHDF的相分离潜力。其次，我将测试这些 突变影响YTHDF在活细胞中的相分离和定位。这些实验将展示 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YTHDFs。第三，我将确定YTHDFs中的这些突变对丰度的影响， 和m6 A mRNA在缺乏内源性YTHDF蛋白表达的细胞中的稳定性。这将测试 具有改变的相分离电位和/或磷酸化位点的YTHDFs影响m6 A mRNA衰变。 总的来说，所提出的实验将测试是否影响YTHDF相分离潜力的突变 和/或在细胞中的定位改变了m6 A mRNA的稳定性，如果这个过程可以被YTHDF调节， 磷酸化这项研究的结果将大大提高我们对这一作用的理解， YTHDF蛋白和相分离影响m6 A mRNA的稳定性。