Regulation of Xist RNA processing in embryonic stem cells

描述(申请人提供)：随着大多数转录组是非编码RNA的发现，具有基因调控功能的非编码RNA开始被视为哺乳动物基因调控的共同特征。鉴于很大一部分基因组编码非编码RNA，人们对这些RNA的调控机制和功能知之甚少。因此，对非编码RNA如何调控以及它们如何调控基因表达的新见解对于全面理解哺乳动物的基因表达是至关重要的。我们以X染色体失活为模型系统来研究非编码RNA的调控和功能。X失活是发育调节的雌性细胞中一条X染色体的转录沉默，用于平衡X连锁基因的剂量与雄性细胞。一对反义的非编码RNA，Xist和TSix RNA，在X-失活的调节中起着中心作用。X-失活是随机的--来自父母双方的X染色体以相同的频率沉默。Xist和tSix RNA在X染色体沉默开始之前表达，是X染色体失活随机发生所必需的。在Xist或TSix突变细胞中，X失活是非随机的，突变和野生型X染色体的命运是固定的。Xist和tSix突变对X染色体的命运有相反的影响：Xist突变的X染色体总是被选择作为活性X，而tSix突变的X染色体总是被选择作为非活性X。Xist和tSix还相互负调控彼此的表达，形成一个反馈环。在这项建议中，我们剖析Xist/TSix反馈环，并探索如何使用它来确保每个X染色体具有相同的沉默频率。 公共卫生相关性：项目叙述(公共卫生相关性)非编码RNA在调节DNA结构、RNA表达和翻译方面发挥关键作用，从而影响正常发育。虽然非编码RNA已经被确定为癌症的标志，并与其他复杂的疾病有关，如冠状动脉疾病和糖尿病，但对它们的调节和功能了解甚少。更好地了解非编码RNA无疑将在这些疾病的诊断和治疗中发挥重要作用。我们以X失活为模型系统来研究非编码RNA的调控和功能。在哺乳动物的雌性细胞中，一条X染色体是沉默的。这种沉默确保了X连锁基因在XX雌性细胞中的剂量与在XY雄性细胞中相同。这一过程对雌性动物的生存至关重要。一对非编码的RNA，Xist和TSix RNA，在X失活的调节中起着中心作用。在本文中，我们建议研究Xist和tSix RNA的调控和功能。我们所学到的无疑将有助于我们对X失活的理解，更广泛地说，有助于非编码RNA的功能。

DESCRIPTION (provided by applicant): ABSTRACT Non-coding RNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation with the discovery that most of the transcriptome is non-coding RNA. Given that a significant proportion of the genome encodes non-coding RNAs, relatively little is known about the regulatory mechanisms and functions of these RNAs. Thus, new insights into how non-coding RNAs are regulated and how they regulate gene expression are essential for a complete understanding of mammalian gene expression. We employ X chromosome inactivation as a model system to study the regulation and function of non-coding RNAs. X-inactivation is the developmentally regulated transcriptional silencing of one X chromosome in female cells, used to equalize X-linked gene dosage with male cells. An antisense pair of non-coding RNAs, Xist and Tsix RNA, are central in the regulation of X-inactivation. X-inactivation is random - the X chromosome from each parent is silenced with equal frequency. Xist and Tsix RNA are expressed before the onset of X chromosome silencing and are necessary for X-inactivation to occur randomly. In Xist or Tsix mutant cells, X-inactivation is non-random and the fates of the mutant and the wild-type X chromosomes are fixed. Xist and Tsix mutations have opposite effects on X chromosome fate: an Xist mutant chromosome is always chosen as the active X and a Tsix mutant X chromosome is always chosen as the inactive X. Xist and Tsix also negatively regulate each other's expression, forming a feedback loop. In this proposal we dissect the Xist/Tsix feedback loop and explore how it is used to ensure that each X chromosome has an equal frequency of being silenced. PUBLIC HEALTH RELEVANCE: Project Narrative (Public Health Relevance) Non-coding RNAs play critical roles in regulating DNA structure, RNA expression, and translation, and thus affect normal development. While non-coding RNAs are already being identified as markers for cancer and associated with other complex diseases such as coronary disease and diabetes, little is understood about their regulation and function. A better understanding of non-coding RNAs will undoubtedly be important in the diagnosis and treatment of these conditions. We use X-inactivation as a model system to study the regulation and function of non-coding RNAs. In mammalian female cells, one X chromosome is silenced. This silencing ensures that X-linked gene dosage in XX female cells is equivalent to that in XY male cells. This process is essential for the survival of females. A pair of non- coding RNAs, Xist and Tsix RNA, is central in the regulation of X-inactivation. In this submission, we propose to study the regulation and function of Xist and Tsix RNA. What we learn will undoubtedly contribute to our understanding of X-inactivation, and more generally to the function of non-coding RNAs.