MicroRNA mediators of stress, dietary restriction and aging

描述（由申请人提供）：衰老和应激反应密切相关，事实上，大多数延长寿命的干预措施似乎至少在一定程度上是通过增强应激反应来实现的。然而，这种中心关系的遗传和机制基础仍然知之甚少。在这里，我们将验证microRNAs （miRNAs）协调应激反应途径以响应延长寿命干预的假设。为了确定秀丽隐杆线虫中连接应激反应与衰老的关键新mirna，我们分析了发育、衰老和应激条件（热休克、饥饿、缺氧和氧化应激）下小rna的表达。由于在应激过程中受到差异调控的mirna可能是应激反应的机制调节剂，因此我们建议表征10种差异表达最多的mirna，并确定它们在应激反应基因调控结构中的位置。我们还将把我们之前对衰老相关mirna的分析和功能分析与这些结果结合起来，以确定与压力和衰老相关的mirna，并测试这些基因是否在这些条件之间提供了机制联系。为了研究microrna在应激和衰老调控中的“网络水平”作用，我们建议阐明参与衰老和应激的基因和microrna的潜在调控网络。为了确定关键的新miRNA和连接这些过程的途径，我们整合了转录因子结合位点信息，与miRNA靶点预测，以建立转录因子、衰老相关miRNA和miRNA生物发生基因之间已知调节关系的初步相互作用网络。我们还建议在miRNA存在和不存在的情况下，通过CLIP-seq和RNA-seq生物化学方法确定关键miRNA的靶点。这些数据将使我们能够改进已知的miRNA-mRNA调控相互作用网络。然后，使用这个网络，我们将发现包含反馈回路和高度连接的相互作用节点的mirna。我们将测试这些高度连接的mirna是否在衰老、应激反应或两者的整合中发挥关键作用。鉴定饮食限制导致寿命延长的miRNA介质。我们的初步数据表明，miR-71和miR-228是连接pha-4和skin -1的关键网络节点，这两种转录因子对饮食限制和其他应激源的反应至关重要。我们将描述这些mirna的作用，并使用我们的基因调控网络来鉴定其他这样的候选mirna。我们还建议通过深度测序来鉴定饮食限制中差异表达的mirna，并将其纳入我们的网络分析中。我们有得天独厚的条件开展这项工作，因为Slack实验室结合了mirna和衰老生物学方面的丰富经验以及全基因组小rna表征方面的领先专业知识。MiRNA类似物和拮抗剂在药理学上是可调控的，因此鉴定秀丽隐杆线虫中关键的衰老和应激反应MiRNA可能直接导致人类寿命和健康延长的干预措施。

DESCRIPTION (provided by applicant): Aging and stress responses are tightly linked~ indeed, most interventions that extend lifespan appear to do so at least in part through potentiation of stress responses. Nevertheless, the genetic and mechanistic basis of this central relationship remains poorly understood. Here we will test the hypothesis that microRNAs (miRNAs) coordinate stress-responsive pathways in response to lifespan-prolonging interventions. We propose the following: To identify critical new miRNAs that link stress responses to aging in C. elegans, we profiled expression of small RNAs during development, aging and in stressed conditions (heat shock, starvation, hypoxia and oxidative stress). Since miRNAs that are differentially regulated during stress are likely to be mechanistic regulators of the stress response, we propose to characterize ten of the most differentially expressed miRNAs and determine their position in the gene-regulatory architecture of stress responses. We will also integrate our previous profiling and functional analyses of aging-associated miRNAs with these results to identify miRNAs that are associated with both stress and aging and test whether these genes provide mechanistic links between these conditions. To investigate "network-level" roles of microRNAs in regulation of stress and aging, we propose to elucidate the underlying regulatory network of genes and miRNAs involved in aging and stress. In order to identify critical new sets of miRNAs and pathways that link these processes, we have integrated transcription-factor binding site information, with miRNA target predictions to build a preliminary interaction network of the known regulatory relationships between transcription factors, aging- associated miRNAs, and miRNA biogenesis genes. We also propose to determine targets of key miRNAs biochemically via CLIP-seq and RNA-seq in the presence and absence of the miRNA. These data will allow us to improve the known miRNA-mRNA regulatory interaction network. Then, using this network, we will find miRNAs that comprise feedback loops and highly connected interaction nodes. We will test whether these highly connected miRNAs play critical roles in aging, stress responses, or in integrating the two. To identify miRNA mediators of lifespan extension due to dietary restriction. Our preliminary data point to miR-71 and miR-228 as key network nodes connected to pha-4 and skn-1, transcription factors critical for the response to dietary restriction and other stressors. We will characterize the roles of thes miRNAs and use our gene- regulatory network to identify other such candidate miRNAs. We also propose to identify miRNAs differentially expressed in dietary restriction via deep sequencing and include these in our network analysis above. We are uniquely well situated to carry out this work, as the Slack lab combines extensive experience in miRNAs and aging biology with leading expertise in genome-wide small-RNA characterization. MiRNA analogues and antagonists are pharmacologically tractable~ thus identifying critical aging and stress responsive miRNAs in C. elegans may lead directly to lifespan and healthspan-prolonging interventions in humans.