Regulation of stress-specific protein translation by the O-GlcNaC transferase ogt-1 and 3' mRNA processing

项目摘要 细胞应激反应在细胞和生物体的生存中起着重要作用，并有助于 广泛的生理过程和人类疾病。的分子结构 大多数应激反应途径都是明确的。一个显著的例外是渗透压 反应，其中动物中的相关压力传感器和信号传导机制很差 明白大多数渗透压反应的研究使用培养的细胞， 复杂性，即细胞外基质、组织机械特性等，无法复制。 为了更好地模拟这些条件，我们研究了活体动物的渗透应激反应， 线虫C.优雅和人类一样，C.线虫对渗透压的反应是代谢 葡萄糖以产生有机渗透剂，例如甘油。我们进行了一个无偏的 遗传筛选以鉴定不表现出渗透剂生物合成基因（Nio）诱导的突变体 基因），并发现了nio-2的多个等位基因，该基因编码唯一的C. elegans同源物 O-GlcNAc转移酶（ogt-1;人体OGT）。OGT后水解O-GlcNAacylates 胞质和核蛋白的Ser/Thr残基，但也表现出重要的GlcNAc酰化 独立的功能。缺乏OGT的哺乳动物细胞不能存活，但C.缺乏ogt的秀丽线虫， 1是可行的和肥沃的，提供了一个独特的机会，研究ogt-1的作用，在细胞 physiology. ogt-1突变体不能在高渗环境中适应和生长， 有机渗透物水平降低，且不诱导渗透物生物合成蛋白GPDH-1。 然而，渗透压物质生物合成基因mRNA的渗透诱导是正常的，这表明， OGT-1在转录后发挥作用。这些缺陷可以通过表达野生型 或无催化活性的人OGT，表明OGT在渗透压中的非典型功能， 应激反应与C. elegans to humans人类.我们还发现了 表型与ogt-1相似的保守3 ′ mRNA加工复合物的相互作用组分。我们 假设ogt-1非典型功能促进应激诱导的mRNA的上调 在渗透胁迫期间，通过与3' RNA加工复合物蛋白相互作用翻译。到 测试这个假设，我们将确定时间，功能和监管要求， ogt-1在渗透胁迫应答中的作用（目的1），明确其特异的基因表达机制 受ogt-1影响（目的2），并确定ogt-1是否调节渗透胁迫反应 通过与3' mRNA切割和多聚腺苷酸化组分的相互作用， 屏幕(Aim 3）。我们的研究将描绘一个新的范式在压力信号和揭示新的 OGT影响细胞生理学的机制。

Project Summary Cellular stress responses play essential roles in cell and organismal survival and contribute to a wide range of physiological processes and diseases in humans. The molecular architecture of most stress response pathways are well defined. A striking exception to this is osmotic stress response, where the relevant stress sensors and signaling mechanisms in animals are poorly understood. Most studies of the osmotic stress response use cultured cells, where in vivo complexities, i.e. the extracellular matrix, tissue mechanical properties, etc., are not replicated. To better mimic these conditions, we study the osmotic stress response in a live animal, the nematode C. elegans. Like humans, C. elegans responds to osmotic stress by metabolizing glucose to produce organic osmolytes, such as glycerol. We performed an unbiased forward genetic screen to identify mutants that exhibit no induction of osmolyte biosynthesis genes (Nio genes) and discovered multiple alleles of nio-2, which encodes the sole C. elegans homolog of the O-GlcNAc transferase (ogt-1; OGT in humans). OGT post-translationally O-GlcNAcylates Ser/Thr residues of cytosolic and nuclear proteins but also exhibits important GlcNAcylation independent functions. Mammalian cells lacking OGT do not survive, but C. elegans lacking ogt- 1 are viable and fertile, providing a unique opportunity to study the role of ogt-1 in cellular physiology. ogt-1 mutants are unable to adapt and grow in hypertonic environments and exhibit reduced organic osmolyte levels and no induction of the osmolyte biosynthesis protein GPDH-1. However, osmotic induction of osmolyte biosynthesis gene mRNAs is normal, suggesting that ogt-1 functions post-transcriptionally. These defects can be rescued by expression of wild type or catalytically inactive human OGT, showing that non-canonical functions of OGT in the osmotic stress response are conserved from C. elegans to humans. We also discovered mutations in interacting components of a conserved 3’ mRNA processing complex that phenocopy ogt-1. We hypothesize that non-canonical functions of ogt-1 facilitate upregulation of stress-induced mRNA translation via interactions with 3’ RNA processing complex proteins during osmotic stress. To test this hypothesis, we will determine the temporal, functional, and regulatory requirements for ogt-1 in the osmotic stress response (Aim 1), identify the specific gene expression mechanism(s) that are affected by ogt-1 (Aim 2), and determine if ogt-1 regulates the osmotic stress response via interactions with 3’ mRNA cleavage and polyadenylation components also identified in our Nio screen. (Aim 3). Our studies will delineate a novel paradigm in stress signaling and reveal new mechanisms by which OGT impacts cell physiology.