Nonsense-Mediated mRNA Decay and Nonsense-Mediated mRNA Decay Factors

这种竞争性更新扩展了我们对无义介导的mRNA衰变（NMD）及其组分的研究。 因子和效应器在人类和小鼠细胞，并自上次竞争性更新，使用小鼠。NMD 通过防止在健康和免疫系统中产生潜在的有毒蛋白质来控制基因表达的质量。 疾病它提供了许多RNA结合蛋白的自动调节，这些蛋白调节前体的剪接， 它们所来源的mRNA。NMD还促进细胞适应不断变化的环境， 约10%的细胞mRNA是天然NMD靶点。目标1建议继续我们令人兴奋的发现， NMD因子的丰度和NMD的效率在缺乏NMD的人神经元细胞中上调。 脆性X智力低下蛋白（FMRP）。FMRP缺乏症是脆性X综合征（FXS）的典型特征， 是智力残疾和自闭症最常见的单基因原因。我们将分析机械和 代表（i）SH-SY 5 Y神经母细胞瘤细胞的神经元分化途径的代谢缺陷，其中 我们已经使用CRISPR-Cas9 n技术敲除了编码FMRP的基因FMR 1，并且（ii）诱导 多能干细胞（iPSC），我们已经从FXS患者成纤维细胞产生。目标2建议， 描述我们发现帽结合蛋白80（CBP 80）直接结合 转录辅激活因子PGC-1α。CBP 80是帽结合异二聚体的一个亚基，其是获得的共- 在蛋白质编码基因的表达过程中转录，我们已经证明，持续通过 在此期间，它在NMD中发挥作用。PGC-1α促进线粒体能量- 产生响应于生理应激的功能，所述生理应激在 衰老和疾病。我们将继续我们的分子研究，以确定如何PGC-1α结合到CBP 80 使用培养的成肌细胞和小鼠骨骼肌再生来控制基因转录， 我们最近使用晶体学和功能测定定义了五种特定的氨基酸变化， PGC-1α的CBP 80结合基序。Aim 3是我们成功筛选新蛋白质的结果 影响NMD的效率，使用稳定的单倍体人细胞的插入基因陷阱诱变， 表达NMD报告基因构建体。我们正在验证特别感兴趣的蛋白质，并正在努力 了解各自对NMD的影响。总之，通过满足以前的目标，我们扩大了 NMD的功能意义，发现NMD在FMRP缺陷中过度活化。我们有 扩大了NMD因素的功能意义，我们发现CBP 80，我们已经表明， 通过外显子连接复合体连接核剪接和细胞质翻译， 翻译也通过与转录辅激活因子的相互作用在早期基因表达步骤中起作用。 我们还在确定国家导弹防御系统效率的新的影响因素。我们的研究结果将为疾病提供新的视角- 相关的细胞代谢。

This competitive renewal extends our studies of nonsense-mediated mRNA decay (NMD) and its constituent factors and effectors in human and mouse cells and, since the last competitive renewal, using mice. NMD controls the quality of gene expression by preventing the production of potentially toxic proteins in health and in disease. It provides for the autoregulation of many RNA-binding proteins that regulate the splicing of the pre- mRNA from which they derive. NMD also promotes the adaptation of cells to changing environments utilizing the ~10% of cellular mRNAs that are natural NMD targets. Aim 1 proposes to pursue our exciting finding that the abundance of NMD factors and the efficiency of NMD are upregulated in human neuronal cells deficient in Fragile X Mental Retardation Protein (FMRP). FMRP deficiency typifies Fragile X Syndrome (FXS), which is the most common single-gene cause of intellectual disability and autism. We will analyze the mechanistic and metabolic defects that typify the neuronal differentiation pathway of (i) SH-SY5Y neuroblastoma cells in which we have knocked-out the FMRP-encoding gene, FMR1, using CRISPR-Cas9n technology, and (ii) induced pluripotent stem cells (iPSCs) that we have generated from FXS-patient fibroblasts. Aim 2 proposes to characterize the functional significance of our finding that cap-binding protein 80 (CBP80) directly binds the transcriptional co-activator PGC-1α. CBP80 is one subunit of the cap-binding heterodimer that is acquired co- transcriptionally during the expression of protein-encoding genes and, we have shown, persists through the pioneer round of translation, during which it functions in NMD. PGC-1α promotes mitochondrial energy- generating functions in response to physiological stresses that become bioenergetically sub-optimal during aging and disease. We will continue our molecular studies to determine how PGC-1α binding to CBP80 controls gene transcription using cultured myoblasts and also skeletal muscle regeneration in mice harboring five specific amino-acid changes in what we recently defined using crystallography and functional assays as the CBP80-binding motif of PGC-1α. Aim 3 results from our successful genetic screen for new proteins influencing the efficiency of NMD using insertional gene-trap mutagenesis of haploid human cells that stably express an NMD reporter construct. We are validating proteins of special interest and are working to understand how each affects NMD. In summary, by satisfying previous aims, we have broadened the functional significance of NMD with the finding that NMD is hyper-activated in FMRP deficiency. We have broadened the functional significance of NMD factors with the finding that CBP80, which we have shown connects nuclear splicing to cytoplasmic translation via exon-junction complexes and the pioneer round of translation, also functions in earlier gene-expression steps via interactions with a transcriptional co-activator. We are also defining new effectors of NMD efficiency. Our results should lend new insight into disease- associated cellular metabolism.