Regulation of Xist RNA processing in embryonic stem cells

摘要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。鉴于基因组中有很大一部分编码非编码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如何被调控以及它们如何调控基因表达的新见解对于全面了解哺乳动物基因表达至关重要。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失活是雌性细胞中一条X染色体的发育调控转录沉默，用于平衡X连锁基因与雄性细胞的剂量。一对反义的非编码RNA， Xist和Tsix RNA，在x -失活的调控中起着核心作用。X染色体失活是随机的——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染色体沉默发生前表达，是X染色体随机失活的必要条件。在Xist或t6突变体细胞中，X失活是非随机的，突变体和野生型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：一条Xist突变染色体总是被选择为活性X，而一条Tsix突变的X染色体总是被选择为非活性X。Xist和Tsix的表达也相互负向调节，形成一个反馈回路。在本提案中，我们剖析了Xist/Tsix反馈回路，并探讨了如何使用它来确保每个X染色体具有相同的沉默频率。

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.