Regulation of RNA Surveillance

描述（由申请人提供）：无义介导的衰变（NMD）是一种质量控制机制，可降解携带提前终止（无义）密码子（PTC）的异常mRNA。三分之一的人类遗传性疾病是由含有无义突变或移码突变的PTC突变基因引起的。最近，NMD也作用于野生型基因的子集变得明显。目前来自几个小组的微阵列分析的估计表明，酿酒酵母、黑腹果蝇和哺乳动物中5%至10%的基因产生受NMD影响的转录本。NMD调节正常转录物水平的能力导致NMD本身受到调节的假设。与这一假设相一致，有证据表明，NMD的大小在不同的细胞类型和不同的发育阶段有所不同。然而，调节NMD的分子和途径尚未阐明。这项建议围绕着最近的两项发现，表明国家导弹防御系统是在负管制控制。首先，发现了一种神经元特异性microRNA（miRNA），它负调控3种在NMD中起作用的因子：RNA解旋酶UPF1、外显子连接复合物（EJC）核心组分MLN51和核糖核酸酶DCP 2。这表明这种miRNA调节NMD本身，这得到了初步数据的支持。这种miRNA在胚胎和出生后的大脑发育过程中表达;在成年人中，它在海马和嗅叶中表达最高。本申请涉及这种miRNA在神经元发育和功能中的作用。还提出了实验来确定这种miRNA通过其靶点UPF1、MLN51和DCP 2控制基因表达的机制。最后，将研究由该miRNA调控的mRNA的全谱。负调控的第二个例子是EJC相关因子UPF3A被它的副产物UPF3B下调。这是一种保守的下调反应，在UPF3B突变的人（其表型是智力迟钝）中被破坏。UPF3A基因可能是在高等真核生物进化过程中被选择的，因为它的副产物UPF3B存在于X染色体上，已知X染色体在雄性生殖细胞减数分裂期间转录失活。在这项提案中，UPF3A在男性生殖细胞和其他细胞类型中的功能得到了解决。还解决了为什么UPF3A表达在具有UPF3B的细胞类型中被特异性抑制。最后，将检查UPF3A抑制的分子机制和从这种抑制中的缓解。这个建议的主要目标是阐明这些负调节回路的潜在机制，并开始理解这种调节的生理后果。通过阐明NMD是如何调控的，将为阐明细胞中的NMD转录网络奠定基础，这反过来可能导致涉及NMD RNA监视途径的疾病的治疗。 无义介导的衰变（NMD）是一种质量控制途径，可以保护细胞免受遗传错误的影响。NMD检测到的遗传错误（那些产生提前终止密码子的错误）导致了三分之一的人类遗传疾病。该提案涉及新发现的负责调节NMD的机制，这些机制与理解正常神经功能和智力迟钝的遗传基础特别相关。

DESCRIPTION (provided by applicant): Nonsense-mediated decay (NMD) is a quality-control mechanism that degrades aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). One-third of human genetic diseases are caused by PTC- containing mutant genes with nonsense or frameshift mutations. Recently, it has become apparent that NMD also acts on a subset of wild-type genes. Current estimates from microarray analyses from several groups indicate that between 5 and 10% of the genes in Saccharomyces cerevisiae, Drosophila melanogaster, and mammals give rise to transcripts that are subject to NMD. The ability of NMD to modulate the level of normal transcripts leads to the hypothesis that NMD is itself subject to regulation. Consistent with this hypothesis, there is evidence that the magnitude of NMD varies in different cell types and at different developmental stages. However, the molecules and pathways that modulate NMD have not been elucidated. This proposal revolves around 2 recent discoveries indicating that NMD is under negative regulatory control. First, a neuron-specific microRNA (miRNA) was discovered that negatively regulates 3 factors that have a role in NMD: the RNA-helicase UPF1, the exon-junction complex (EJC) core component MLN51, and the ribonuclease DCP2. This suggests that this miRNA regulates NMD itself, which is supported by preliminary data. This miRNA is expressed during both embryonic and postnatal development of the brain; in the adult, it is most highly expressed in the hippocampus and olfactory lobe. This application addresses the role of this miRNA in neuronal development and function. Experiments are also proposed to determine the mechanisms by which this miRNA controls gene expression through its targets UPF1, MLN51, and DCP2. Finally, the full spectrum of mRNAs regulated by this miRNA will be investigated. The second example of negative regulatory control is that the EJC-associated factor UPF3A is downregulated by its paralog UPF3B. This is a conserved downregulatory response that is disrupted in humans with UPF3B mutations (whose phenotype is mental retardation). The UPF3A gene may have been selected for during the evolution of higher eukaryotes because its paralog, UPF3B, is present on the X chromosome, which is known to be transcriptionally inactivated during meiosis in male germ cells. In this proposal, the function of UPF3A in male germ cells and other cell types is addressed. Also addressed is why UPF3A expression is specifically suppressed in cell types that have UPF3B. Finally, the molecular mechanism of UPF3A suppression and relief from this suppression will be examined. The broad goals of this proposal are to elucidate the underlying mechanism for these negative regulatory circuits and to begin to understand the physiological consequences of this regulation. By elucidating how NMD is regulated, a foundation will be made for elucidating the NMD transcriptional networks in cells, which in turn may lead to therapies for diseases involving the NMD RNA surveillance pathway. PUBLIC HEALTH RELEVANCE: Nonsense-mediated decay (NMD) is a quality-control pathway that protects cells from genetic mistakes. The genetic mistakes detected by NMD (those that generate premature termination codons) are responsible for one-third of human genetic diseases. This proposal addresses newly discovered mechanisms responsible for regulating NMD that are particularly relevant for understanding normal neurological function and the genetic basis for mental retardation.