Post-transcriptional mechanisms of gene regulation in cardiac cell growth and development

一、摘要 心脏病仍然是美国的主要死亡原因。尽管我们知道 与心脏病相关的危险因素，其分子机制在很大程度上仍不清楚。健康的人 成人心脏与其他组织的独特之处在于，蛋白质合成的速度明显低于大多数组织 组织和低于发育中的心脏。然而，在心肌肥厚期间，翻译率增加。 这表明了一种组织特异性的机制来调节哺乳动物心脏的翻译率。我们的实验室有 确认了心肌细胞在发育过程中总蛋白质合成减少的机制 通过缩短Poly(A)尾巴，导致闭合环模型中多聚体形成的减少 翻译的问题。这一调节在生理性和病理性肥厚期间都被逆转，当 心肌细胞的翻译需求增加。此外，我们还发现，核多聚(A)结合 蛋白(PABPN1)在哺乳动物成年心肌和骨骼肌中转录后沉默，但它 在病理性心肌肥厚中重新表达。PABPN1是一种选择性多聚腺苷酸化的调节剂 (APA)和Poly(A)尾长，这两者都会影响转录本的翻译。我们的中心假设 PABPN1在心肌细胞中受到动态调节，以调节翻译速率和适应生长需求 通过多聚腺苷依赖的机制。这项提议的目的是阐明 PABPN1在心脏发育和生长中的作用(S)，并确定在此过程中PABPN1是如何调节的 条件。AIMS 1和2将使用有条件的PABPN1基因敲除和过度表达的小鼠来确定 PABPN1在心脏发育和肥大中的生理作用 PABPN1活性及其在确定心脏特异基因表达程序中的作用。在目标3中，我们将使用 超分辨显微镜、CRISPR-Cas9介导的基因组编辑和RNA反义寡核苷酸下拉 识别调控机制的方法(S)和转录后沉默PABPN1的因素 在心脏发育过程中。

I. ABSTRACT Heart disease remains the leading cause of death in the United States. Despite what we know about the risk factors associated with heart disease, the molecular mechanisms are still largely unknown. The healthy adult heart is unique from other tissues in that the rate of protein synthesis is dramatically lower than in most tissues and lower than the developing heart. However, during cardiac hypertrophy, translation rates increase. This suggests a tissue-specific mechanism for regulating translation rates in the mammalian heart. Our lab has identified a mechanism by which total protein synthesis in cardiomyocytes is decreased during development through shortening of poly(A) tails, leading to a decrease in polysome formation through the closed-loop model of translation. This regulation is reversed during both physiologic and pathologic hypertrophy when the translation needs of cardiomyocytes are increased. Also, we have discovered that the nuclear poly(A) binding protein (PABPN1) is post-transcriptionally silenced in mammalian adult cardiac and skeletal muscle but it becomes re-expressed in pathologic cardiac hypertrophy. PABPN1 is a regulator of alternative polyadenylation (APA) and poly(A) tail length, both of which can influence the translation of transcripts. Our central hypothesis is that PABPN1 is dynamically regulated in cardiac myocytes to tune translation rates and suite growth needs through a polyadenylation dependent mechanism. The objective of this proposal is to elucidate the exact function(s) of PABPN1 in cardiac development and growth and identify how PABPN1 is regulated during these conditions. Aims 1 and 2 will use conditional PABPN1-knockout and overexpressing mice to determine the physiologic roles of PABPN1 in cardiac development and hypertrophy while defining the molecular basis of PABPN1 activity and its role in determining cardiac-specific gene expression programs. In Aim 3, we will use super-resolution microscopy, CRISPR-Cas9 mediated genome editing, and RNA antisense-oligo pulldown approaches to identify the regulatory mechanism(s) and factors that post-transcriptionally silence PABPN1 during cardiac development.