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

心肌细胞生长发育中基因调控的转录后机制

• 批准号: 10221031
• 负责人: Auinash Kalsotra
• 金额: $ 38.56万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221031
• 关键词: Adult; Affect; Antisense Oligonucleotides; Antisense RNA; Binding Sites; Biological Assay; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiac development; Cause of Death; Cell Nucleus; Cleaved cell; Cues; Cytoplasm; Development; Disease; Funding; Gene Expression; Gene Expression Regulation; Gene Silencing; Genetic; Genetic Transcription; Goals; Growth; Growth and Development function; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Hypertrophy; Knock-out; Knockout Mice; Laboratories; Length; Mediating; Messenger RNA; Microscopy; Modeling; Molecular; Mus; Muscle; Muscle Cells; Myocardial; Myocardial Infarction; Myocardium; Neonatal; Nuclear; Outcome; Pathologic; Performance; Physiological; Poly A; Poly(A) Tail; Poly(A)-Binding Proteins; Polyadenylation; Polyribosomes; Post-Transcriptional Regulation; Protein Biosynthesis; RNA; RNA Splicing; Regulation; Regulatory Pathway; Resolution; Risk Factors; Role; Signal Transduction; Skeletal Muscle; Stimulus; Stress; Tamoxifen; Testing; Tetracyclines; Therapeutic; Tissues; Trans-Activators; Transcript; Transgenic Mice; Translating; Translations; United States; Work; cardiogenesis; cell growth; experimental study; fetal; genome editing; heart function; hemodynamics; in vivo; insight; mRNA Export; mouse model; overexpression; polysome profiling; postnatal; postnatal development; premature; programs; protein expression; response; single molecule; transcription termination; transcriptome; transcriptome sequencing; translatome

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.

I.摘要 心脏病仍然是美国的主要死因。尽管我们知道 与心脏病相关的危险因素，分子机制仍然在很大程度上未知。健康 成人心脏与其他组织不同，其蛋白质合成速率显著低于大多数心脏。 低于发育中的心脏。然而，在心脏肥大期间，翻译速率增加。 这表明在哺乳动物心脏中调节翻译速率的组织特异性机制。我们的实验室 确定了一种机制，通过这种机制，心肌细胞中的总蛋白质合成在发育过程中减少 通过缩短poly（A）尾，通过闭环模型导致多聚核糖体形成减少 的翻译。这种调节在生理性和病理性肥大期间被逆转， 心肌细胞的翻译需求增加。此外，我们还发现， 蛋白（PABPN 1）在哺乳动物成体心肌和骨骼肌中转录后沉默，但它 在病理性心脏肥大中重新表达。PABPN 1是选择性多聚腺苷酸化的调节因子 (APA)和poly（A）尾长，这两者都可以影响转录物的翻译。我们的核心假设 PABPN 1在心肌细胞中受到动态调节，以调节翻译速率并适应生长需要， 通过多聚腺苷酸化依赖机制。本提案的目的是阐明 PABPN 1在心脏发育和生长中的功能，并确定PABPN 1在这些过程中如何调节 条件目的1和2将使用条件性PABPN 1敲除和过表达小鼠来确定PABPN 1的表达。 PABPN 1在心脏发育和肥大中的生理作用，同时定义了 PABPN 1活性及其在确定心脏特异性基因表达程序中的作用在目标3中，我们将使用 超分辨率显微镜、CRISPR-Cas9介导的基因组编辑和RNA反义寡核苷酸下拉 鉴定转录后沉默PABPN 1的调节机制和因子的方法 在心脏发育过程中。