PAX9, ribosome biogenesis, and congenital disease

项目总结/摘要 背景和中心假设：核糖体病是由制造蛋白质的过程中断引起的。 核糖体，负责合成所有细胞蛋白质的复杂分子机器。虽然有人可能 期望在这样一个基本过程中的缺陷导致一个不可活的有机体，这种疾病反而表现在 组织特异性体征和症状。在核糖体病Treacher柯林斯综合征中，这种组织特异性 表现为由压力敏感神经嵴细胞中的核仁压力反应引起的颅面缺陷。 配对盒9（PAX 9）基因的突变会导致六颗以上的恒牙缺失，或少牙症， 以及颅面畸形和脱发。这些症状与核糖体病相似， 特雷彻柯林斯综合征。在人MCF 10A细胞中筛选基因组范围的siRNA，以获得所需的蛋白质， 核糖体的产生揭示了RNA聚合酶II（POL II）转录因子的意想不到的作用， PAX 9，在人类核糖体生物合成中。初步结果表明，PAX 9缺失导致了细胞内的缺陷。 前核糖体RNA（pre-rRNA）加工以及减少一个基因子集的转录， 核仁蛋白我推测PAX 9通过促进核糖体的合成间接改变了哺乳动物核糖体的生物合成。 这些基因中的一个或多个的转录，这些基因是在细胞核仁中制造核糖体所必需的。我建议 在PAX 9突变的人中观察到的颅面畸形和少齿症与PAX 9的 在核糖体生物合成中的重要作用。具体目标：具体目标1将确认需要PAX 9蛋白 用于转录编码核仁蛋白的候选基因。我会确认北方的污点 表型与候选者的PAX 9转录直接相关。我还将证明PAX 9结合 直接作用于候选基因并调节其转录。具体目标2将测试 发育中胚胎中PAX 9缺失或突变将颅面畸形与核糖体联系起来 生物起源首先，我将建立热带爪蟾作为研究PAX 9对两者影响的模型系统。 颅面发育和核糖体生物发生。然后，我将测试候选基因在多大程度上 特异性目标1概括了PAX 9耗尽后观察到的缺陷。

PROJECT SUMMARY/ABSTRACT BACKGROUND AND CENTRAL HYPOTHESIS: Ribosomopathies are caused by disruptions in making ribosomes, the intricate molecular machines responsible for synthesizing all cellular proteins. While one might expect defects in such an essential process to result in an inviable organism, such disorders instead manifest in tissue-specific signs and symptoms. In the ribosomopathy Treacher Collins syndrome, this tissue specificity manifests in craniofacial defects arising from the nucleolar stress response in stress-sensitive neural crest cells. Mutations in the Paired box 9 (PAX9) gene result in the loss of more than six permanent teeth, or oligodontia, as well as craniofacial dysmorphology and hair loss. These symptoms are similar to those of the ribosomopathy, Treacher Collins syndrome. A genome-wide siRNA screen in human MCF10A cells for proteins required for the production of ribosomes revealed an unexpected role for the RNA Polymerase II (POL II) transcription factor, PAX9, in human ribosome biogenesis. Preliminary results have shown that PAX9 depletion results in defects in pre-ribosomal RNA (pre-rRNA) processing as well as in decreased transcription of a subset of genes that encode nucleolar proteins. I hypothesize that PAX9 alters mammalian ribosome biogenesis indirectly by promoting the transcription of one or more of these genes required for making ribosomes in the cell nucleolus. I propose that the craniofacial dysmorphology and oligodontia seen in humans with PAX9 mutations is connected to PAX9’s crucial role in ribosome biogenesis. SPECIFIC AIMS: Specific Aim 1 will confirm that the PAX9 protein is required for the transcription of candidate genes encoding nucleolar proteins. I will confirm that the northern blot phenotype is directly related to PAX9’s transcription of the candidates. I will also demonstrate that PAX9 binds directly to and regulates the transcription of the candidate genes. Specific Aim 2 will test the extent to which PAX9 depletion or mutation in a developing embryo connects craniofacial dysmorphology to ribosome biogenesis. First, I will establish Xenopus tropicalis as a model system for studying the effects of PAX9 on both craniofacial development and ribosome biogenesis. I will then test the extent to which the candidate genes from Specific Aim 1 recapitulate the defects seen upon PAX9 depletion.