MicroRNA mediators of stress, dietary restriction and aging

描述（由申请人提供）：衰老和压力反应是紧密相连的，事实上，大多数延长寿命的干预措施似乎至少部分是通过增强压力反应来实现的。然而，这种核心关系的遗传和机制基础仍然知之甚少。在这里，我们将测试的假设，即microRNAs（miRNAs）协调应激反应途径，以响应延长寿命的干预措施。我们提出以下建议：鉴定关键的新的miRNAs，将应激反应与衰老联系起来。在线虫中，我们分析了小RNA在发育、衰老和应激条件（热休克、饥饿、缺氧和氧化应激）中的表达。由于在应激过程中差异调节的miRNA可能是应激反应的机制调节剂，我们建议表征10个最差异表达的miRNA，并确定它们在应激反应基因调控结构中的位置。我们还将将之前对衰老相关miRNA的分析和功能分析与这些结果相结合，以识别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，我们提出阐明衰老和应激相关基因和miRNAs的潜在调控网络。为了鉴定关键的新的miRNA组和连接这些过程的通路，我们整合了转录因子结合位点信息，与miRNA靶预测一起构建了转录因子、衰老相关miRNA和miRNA生物发生基因之间已知调控关系的初步相互作用网络。我们还建议在存在和不存在miRNA的情况下通过CLIP-seq和RNA-seq生物化学地确定关键miRNA的靶标。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，我们将找到包含反馈环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miRNAs是否在衰老，应激反应或整合两者中发挥关键作用。鉴定饮食限制导致寿命延长的miRNA介导物。我们的初步数据表明，miR-71和miR-228是连接到pha-4和skn-1的关键网络节点，这两个转录因子对饮食限制和其他压力源的反应至关重要。我们将描述这些miRNAs的作用，并利用我们的基因调控网络来鉴定其他类似的候选miRNAs。我们还建议通过深度测序来鉴定在饮食限制中差异表达的miRNA，并将这些miRNA包括在我们上面的网络分析中。我们处于开展这项工作的独特位置，因为Slack实验室将miRNA和衰老生物学方面的丰富经验与全基因组小RNA表征方面的领先专业知识相结合。miRNA的类似物和拮抗剂是易处理的，从而鉴定了C. elegans可能直接导致延长人类寿命和健康的干预措施。

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.