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有机体的生存中起着至关重要的作用，并有助于 人类的各种生理过程和疾病。分子的构型 大多数应激反应途径都有明确的定义。其中一个显著的例外是渗透压力。 响应，动物体内相关的应激传感器和信号机制很差 明白了。大多数渗透应激反应的研究使用培养细胞，在体内 复杂性，即细胞外基质、组织机械性能等，不被复制。 为了更好地模拟这些情况，我们研究了活体动物的渗透应激反应 线虫线虫。和人类一样，线虫对渗透胁迫的反应是通过代谢 葡萄糖产生有机渗透物质，如甘油。我们表演了一个不偏不倚的前锋 基因筛选以鉴定不诱导渗透调节物质生物合成基因(Nio)的突变体 基因)，并发现了NiO-2的多个等位基因，它编码唯一的线虫同源物 O-GlcNAc转移酶(OGT-1；人的OGT)。OGT翻译后O-GlcNacylates 胞浆和核蛋白的Ser/Thr残基，但也具有重要的GlcN酰化作用 独立的功能。缺乏OGT的哺乳动物细胞无法存活，但缺乏OGT的线虫- 1为研究OGT-1在细胞中的作用提供了一个独特的机会 生理学。OGT-1突变体不能适应和生长在高渗环境中并表现出 降低有机渗透压水平，不诱导渗透压生物合成蛋白GPDH-1。 然而，渗透诱导渗透压生物合成基因mRNAs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来补救 或催化失活的人OGT，表明OGT在渗透压中的非正则功能 从线虫到人类，应激反应都是保守的。我们还发现了基因突变 具有OGT-1表型的保守的3‘mRNA加工复合体的相互作用组分。我们 假设OGT-1的非正则功能促进应激诱导的mRNA上调 在渗透胁迫下，通过与3‘RNA加工复杂蛋白质的相互作用进行翻译。至 测试这一假设，我们将确定时间、功能和法规要求 OGT-1在渗透胁迫反应中的作用(目标1)，确定特定的基因表达机制(S) 并确定OGT-1是否调节渗透应激反应 通过与3‘mRNA裂解和多腺苷化成分的相互作用，在我们的NiO中也发现了 屏幕上。(目标3)。我们的研究将描绘一种压力信号的新范式，并揭示新的 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.