MicroRNA mediators of stress, dietary restriction and aging

描述(申请人提供)：衰老和应激反应紧密相连~事实上，大多数延长寿命的干预措施似乎至少部分是通过加强应激反应来实现的。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，我们将检验这一假设，即microRNAs(MiRNAs)协调应激反应通路，以响应延长寿命的干预。我们提出如下建议：为了确定线虫中将应激反应与衰老联系起来的关键新miRNAs，我们分析了小RNA在发育、衰老和应激条件(热休克、饥饿、低氧和氧化应激)中的表达。由于在应激过程中差异调控的miRNAs可能是应激反应的机械性调节者，我们建议对十个差异表达最大的miRNAs进行鉴定，并确定它们在应激反应的基因调控体系中的位置。我们还将把我们之前对衰老相关miRNAs的分析和功能分析与这些结果结合起来，以识别与应激和衰老相关的miRNAs，并测试这些基因是否提供了这些条件之间的机械联系。为了研究microRNAs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miRNAs的潜在调控网络。为了识别关键的新的miRNAs集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和miRNA靶标预测，以建立一个初步的相互作用网络，该网络包含转录因子、衰老相关的miRNAs和miRNA生物发生基因之间的已知调控关系。我们还建议在存在和不存在miRNA的情况下，通过CLIP-SEQ和RNA-SEQ以生化方式确定关键miRNAs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，我们将找到包括反馈环和高度连接的交互节点的miRNAs。我们将测试这些高度连接的miRNAs是在衰老、应激反应中发挥关键作用，还是在整合两者中发挥关键作用。鉴定由于饮食限制而延长寿命的miRNA介体。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应激源的反应至关重要。我们将表征这些miRNAs的作用，并使用我们的基因调控网络来识别其他这样的候选miRNAs。我们还建议通过深度测序来识别在饮食限制中差异表达的miRNAs，并将其包括在我们上面的网络分析中。我们得天独厚地开展这项工作，因为Slack实验室将miRNAs和衰老生物学方面的丰富经验与基因组范围小RNA表征方面的领先专业知识相结合。MiRNA类似物和拮抗剂在药理上是容易处理的，因此识别线虫中关键的衰老和应激反应miRNA可能直接导致人类的寿命和健康延长干预。

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.