Deciphering the role of a circadian lncRNA in cardiac remodeling

项目摘要 心力衰竭(HF)与50%的5年死亡率有关，而且发病率仍在上升，大多数 与缺血性心肌病相关的4例。确定对抗心脏病理的新策略 心肌梗死(MI)后重构是临床迫切需要。长的非编码RNA(LncRNA)具有 最近作为心脏发育和疾病的调节者而出现。然而，大多数LncRNA的功能 仍然不为人知。 我们已经确定了第一个心脏特异的昼夜节律，它影响心脏重构。 通过调节可选剪接来实现MI后。为了更好地了解Circa及其受体的分子机制 对于心脏中的伙伴蛋白，我们提出了以下具体目标：1.鉴定 CircA核定位和与剪接体相互作用所必需的。我们将使用最先进的化学品 用探针技术确定CircA的二级结构，然后利用结构突变鉴定其功能 图案。2.确定Circa是否在反式中起作用，其功能是否依赖于其振荡 表情。我们将通过一系列的“救援”实验来明确地确定Circa的功能 在我们创造的小说中，敲除老鼠。时间限制表达式将用于测试 摇摆的表情。3.确定CircA影响心脏剪接的分子基础。我们的预赛 数据表明，Circa可能会抑制剪接调控因子hnRNPA1。我们将测试它们之间的功能交互 Circa和hnRNPA1使用我们鉴定的心脏靶基因。我们将进一步研究hnRNAPA1的作用 通过使用eCLIPseq/RNAseq识别其剪接靶点，并测试其在MI后重塑中的作用 HnRNPA1的体外和体内实验。这一建议的完成将对理解剪接产生重大影响 心肌梗死后病理重塑过程中的心脏调节和潜在的扩展治疗策略 接受心衰治疗。

Project Summary Heart failure (HF) is associated with a 5-year mortality of 50% and the incidence is still rising with most cases associated with ischemic cardiomyopathy. Identifying novel strategies to counteract cardiac pathological remodeling post myocardial infarction (MI) is of urgent clinical need. Long non-coding RNAs (lncRNA) have recently emerged as regulators of cardiac development and disease. However, the function of most lncRNAs remains unknown. We have characterized the first cardiac specific circadian lncRNA Circa, which affects cardiac remodeling post MI through regulating alternative splicing. To better understand the molecular mechanisms of Circa and its partner proteins in the heart, we propose the following specific aims: 1. Identify the RNA structures that are required for Circa nuclear localization and interaction with spliceosome. We will use the state-of-the-art chemical probing technique to define the secondary structure of Circa, then use structure mutants to identify its functional motifs. 2. Determine whether Circa acts in trans and whether its function is dependent on its oscillatory expression. We will unambiguously determine Circa function as a transcript by a series of “rescue” experiments in the novel knock out mice we created. Time restricted expression will be used to test the necessity of the oscillatory expression. 3. Define the molecular basis by which Circa affects splicing in the heart. Our preliminary data suggests Circa may suppress splicing regulator hnRNPA1. We will test the functional interaction between Circa and hnRNPA1 using a cardiac target gene we identified. We will further investigate the role of hnRNAPA1 in the post MI remodeling by identifying its splicing targets using eCLIPseq/RNAseq and test the function of hnRNPA1 in vitro and in vivo. Completion of this proposal will have significant impact in understanding splicing regulation in the heart during post MI pathological remodeling and potentially expand our therapeutic strategies for HF treatment.