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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 N6-甲基腺苷 (m6a) 是转录组中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 mRNA 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：YTH 结构域，结合 m6A 和一个低复杂性结构域，富含促进疾病的氨基酸。低复杂度域允许 YTHDF 蛋白发生“相分离”，这是一种富含蛋白质和 RNA 的液滴形成于细胞质中。通过与富含 m6A 的 mRNA 形成多价相互作用，YTHDF 介导的相分离允许将 m6A-mRNA 隔离在细胞中的相分离液滴中。然而，它并不是知道如何控制 YTHDF 蛋白的相分离电位，或者该过程是否有助于 m6A mRNA 不稳定。对 YTHDF 蛋白的低复杂性结构域的分析揭示了几种朊病毒样域和假定的磷酸化位点。在本提案中，我寻求解决以下关键问题：如何 YTHDF 的类朊病毒结构域和磷酸化控制相分离吗？ m6A mRNA 降解是否需要分离？为了探索这个问题，我将首先确定突变是如何发生的 YTHDF 蛋白的低复杂性结构域和假定的磷酸化位点影响体外相分离潜在的。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 中的这些突变对丰度的影响以及缺乏内源性 YTHDF 蛋白表达的细胞中 m6A mRNA 的稳定性。这将测试是否具有改变的相分离电位和/或磷酸化位点的 YTHDF 会影响 m6A mRNA 的衰减。总的来说，拟议的实验将测试是否影响 YTHDF 相分离电位的突变和/或细胞中的定位会改变 m6A mRNA 的稳定性，并且该过程是否可以通过 YTHDF 调节磷酸化。这项研究的结果将大大提高我们对 YTHDF 蛋白和相分离在影响 m6A mRNA 稳定性方面发挥着作用。