Non-coding RNAs in Transcription and Chromatin Architecture

描述(由申请人提供)： 我们最近发现了一类被称为ncRNA激活(ncRNA-a)的长ncRNA，它可以在很长的距离上激活邻近的蛋白质编码基因的表达。我们目前的数据表明，ncRNA-a通过与共激活复合体Mediator结合来促进DNA环化。将研究ncRNA-a和介体复合体之间的联系，以了解靶标选择的特异性是如何实现的，并确定RNA对转录激活的贡献。这三个目的是为了提供对激活的ncRNAs及其与介体复合体的关联的彻底的分子理解。AIM1描述了检查ncRNA的分子基础的实验-a核定位和转录激活。目的2描述描述ncRNA-a的分子基础和介体复合体相互作用的实验。我们将确定ncRNA-a是否对Mediator的靶点招募起关键作用，并进行结构/功能实验，以评估ncRNA-a/Mediator相互作用在转录激活中的重要性。AIM3将评估ncRNA-a激活CDK8激酶活性的分子基础。重要的是，对激酶活性至关重要的介体复合体MED12亚单位的反复突变会导致多种人类认知综合征和良性平滑肌肿瘤的发展。因此，我们将验证这样的假设，即这些突变通过破坏其关联来影响介体的激酶活性 与核糖核酸-a结合。

DESCRIPTION (provided by applicant): Project Summary We have recently identified a class of long ncRNA termed ncRNA-activating (ncRNA-a), which can activate the expression of neighboring protein-coding genes at long distances. Our current data suggest that ncRNA-a function by associating with the co-activator complex, Mediator, to promote DNA looping. The association between ncRNA-a and the Mediator complex will be investigated to understand how specificity for target selection is achieved and to pinpoint the contribution of RNA to transcriptional activation. The three Aims are designed to provide a thorough molecular understanding of the activating ncRNAs and their association with the Mediator complex. Aim1 describes experiments that examine the molecular basis for ncRNA-a nuclear localization and transcriptional activation. Aim 2 describes experiments to delineate the molecular basis of ncRNA-a and the Mediator complex interaction. We will determine whether ncRNA-a is critical for the recruitment of Mediator to its target sites and perform structure/function experiments to assess the importance of ncRNA-a/Mediator interaction in transcriptional activation. Aim3 will assess the molecular basis by which ncRNA-a activates the CDK8 kinase activity. Importantly, recurrent mutations in Med12 subunit of Mediator complex, which is critical for kinase activity, result in development of multiple human cognitive syndromes and a benign smooth muscle neoplasm. Therefore, we will test the hypothesis that such mutations affect the kinase activity of Mediator by disrupting its association with the ncRNA-a.