YTHDF3 as a critical regulator of cardiac function

项目摘要 作为全球主要的死亡原因，心力衰竭是研究人员寻求发现的主要挑战 可以拯救无数生命的疗法心脏损伤后，心脏开始重塑自己的方式， 最初是适应性的，但随着时间的推移，这种先天的应对机制可能会加速心力衰竭的发作。阐明 从适应性心脏肥厚性重构到心力衰竭的进展机制将 对发现这种致命疾病的新疗法产生了巨大影响。虽然基因调控 通过信使RNA（mRNA）转录的表达已经被广泛研究，直到最近， 已经出现了对可能发生在mRNA上的化学修饰的重要性的认识。这项建议 重点关注mRNA的N6-腺苷（m6 A）的甲基化，其是最丰富的内部mRNA 真核生物中的修饰。我们实验室以前的研究表明，在细胞中调节m6 A含量， 心脏的压力足以驱动心脏重塑并影响心脏对压力的反应能力。尽管 这种情况发生的确切机制尚不清楚。m6 A修饰的mRNA的命运 由YTH结构域家族（YTHDF）成员调控。我们发现YTHDF 3特别重要， 在心肌细胞中，它定位于细胞核并与肌细胞增强因子2D（MEF 2D）结合， 是调节肥大性心脏生长的重要转录因子。此外，我们发现， YTHDF 3减轻了压力过载损伤后的病理性重塑。根据这些初步数据， 我们推测YTHDF 3通过调节心肌细胞的大小和应激诱导的重构， 由MEF 2D转录的m6 A修饰的mRNA的加工。为了验证这个假设，我们已经 产生并验证了一种新的小鼠品系，其中YTHDF 3可以在心肌细胞中选择性缺失 （YTHDF3-cKO）。在目标1中，我们将研究YTHDF 3在基线和应激小鼠心脏中的作用 使用纵向超声心动图分析，并评估病理的组织学和分子学体征， 终点时间点。在目标2中，我们将确定YTHDF 3调节细胞命运的机制。 心肌细胞中MEF 2D转录的m6 A-mRNA的特定子集。首先，我们将进一步描述 通过定义所涉及的相应结构域来确定YTHDF 3和MEF 2D之间的结合。然后，我们将解剖 YTHDF 3与MEF 2D mRNA靶点的结合，并决定这些靶点的随后稳定性、输出和翻译。 成绩单最后，在目标3中，我们将采取公正的方法，在全球范围内调查 YTHDF 3通过交联调节健康和应激的成年心肌细胞中的mRNA生物学 YTHDF 3结合的mRNA的免疫沉淀，随后测序（CLIP-seq）。我们的方法是创新的 而且意义重大，因为它将是第一个定义YTHDF 3在心脏中作用的项目，这可能会导致一个新的 基于mRNA甲基化生物学的治疗学领域。

PROJECT SUMMARY As the global leading cause of death, heart failure is a major challenge for researchers in their quest to discover therapeutics that can save countless lives. After cardiac injury, the heart begins to remodel itself in a way that is initially adaptive, but this innate coping mechanism may over time expedite heart failure onset. Elucidating the mechanisms which underly the progression from adaptive cardiac hypertrophic remodeling to heart failure will dramatically impact the discovery of novel therapeutics for this deadly disease. While regulation of gene expression through transcription of messenger RNA (mRNA) has been extensively studied, only recently an appreciation for the importance of chemical modifications that can occur on mRNA has emerged. This proposal focuses on the methylation of the N6-Adenosine of mRNA (m6A), which is the most abundant internal mRNA modification in eukaryotes. Previous research from our lab has shown that modulation of m6A content in the heart is sufficient to drive cardiac remodeling and to affect the ability of the heart to respond to stress. Despite this, the exact mechanisms through which this occurs is not well understood. The fate of m6A-modified mRNAs is regulated by members of the YTH Domain Family (YTHDF). We found that YTHDF3 is specifically important in cardiomyocytes, where it localizes to the nucleus and binds to Myocyte Enhancer Factor 2D (MEF2D), which is an important transcription factor regulating hypertrophic cardiac growth. Further, we have found that knockout of YTHDF3 mitigates pathological remodeling following pressure overload injury. Given these preliminary data, we hypothesize that YTHDF3 regulates cardiomyocyte size and stress-induced remodeling by modulating the processing of m6A-modified mRNAs transcribed by MEF2D. To test this hypothesis, we already generated and validated a new mouse line in which YTHDF3 can be selectively deleted in cardiomyocytes (YTHDF3-cKO). In Aim 1, we will investigate the role of YTHDF3 at baseline and in the stressed murine heart using longitudinal echocardiography analysis, and assessing histological and molecular signs of pathology at the terminal time point. In Aim 2, we will determine the mechanism through which YTHDF3 regulates the fate of specific subsets of MEF2D-transcribed m6A-mRNAs in cardiomyocytes. First, we will further characterize the binding between YTHDF3 and MEF2D by defining the respective domains involved. Then, we will dissect the binding of YTHDF3 to MEF2D mRNA targets and determine consequent stability, export, and translation of these transcripts. Finally, in Aim 3, we will undertake an unbiased approach to more globally investigate the role of YTHDF3 in regulating mRNA biology in healthy and stressed adult cardiomyocytes by cross-linking immunoprecipitations of YTHDF3-bound mRNAs followed by sequencing (CLIP-seq). Our approach is innovative and significant, as it will be the first project to define the role of YTHDF3 in the heart, which may lead to a new field of therapeutics based on the biology of mRNA methylation.