Deciphering the role of a circadian lncRNA in cardiac remodeling

项目摘要 心力衰竭（HF）与50%的5年死亡率相关，并且随着大多数患者的死亡， 与缺血性心肌病相关的病例。确定对抗心脏病理性疾病的新策略 心肌梗死（MI）后的重构是临床上迫切需要的。长链非编码RNA（lncRNA） 作为心脏发育和疾病的调节因子。然而，大多数lncRNA的功能 仍然未知。 我们已经描述了第一个心脏特异性昼夜节律lncRNA Circa，它影响心脏重塑 通过调节选择性剪接来表达。为了更好地了解Circa及其分子机制， 伴侣蛋白在心脏，我们提出以下具体目标：1。识别RNA结构， 所需的Circa核定位和相互作用的剪接体。我们会用最先进的化学物质 利用探针技术确定Circa的二级结构，然后利用结构突变体鉴定其功能 图案2.确定Circa是否反式作用，以及其功能是否依赖于其振荡 表情我们将通过一系列的“拯救”实验明确地确定Circa作为转录本的功能 在小说中我们创造了敲除老鼠。时间限制表达式将用于测试 振荡表达式3.定义Circa影响心脏剪接的分子基础。我们的初步 数据表明Circa可能抑制剪接调节因子hnRNPA 1。我们将测试功能之间的相互作用 Circa和hnRNPA 1使用我们鉴定的心脏靶基因。我们将进一步研究hnRNAPA 1的作用， 通过使用eCLIPseq/RNAseq鉴定其剪接靶点， hnRNPA 1在体外和体内。完成本提案将对理解拼接产生重大影响 在心肌梗死后病理性重塑过程中的心脏调节，并可能扩展我们的治疗策略 HF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.