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

O-GlcNaC 转移酶 ogt-1 和 3 mRNA 加工对应激特异性蛋白翻译的调节

基本信息

批准号：
10663299
负责人：
SAMUEL T LAMITINA
金额：
$ 34.68万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-09 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10663299
关键词：
Affect Alleles Anabolism Animals Architecture Binding Biochemical Biological Models Caenorhabditis elegans Cell Culture Techniques Cell Nucleus Cell physiology Cells Cellular Stress Complex Cultured Cells Defect Dehydration Development Diabetes Mellitus Disease Environment Enzymes Exhibits Extracellular Matrix Gene Expression Genes Genetic Genetic Screening Genomic approach Glucose Glycerol Glycerol-3-Phosphate Dehydrogenase Growth Health Homologous Gene Human Link Mammalian Cell Mammals Mediating Messenger RNA Molecular Mutation Nematoda Nerve Degeneration Nuclear Nuclear Export Nuclear Proteins O-GlcNAc transferase Organism Osmosis Pathway Analysis Pathway interactions Phenocopy Phenotype Phosphorylation Phosphotransferases Physiological Physiological Processes Play Poly A Polyadenylation Post-Translational Protein Processing Process Protein Biosynthesis Proteins RNA RNA Processing Regulation Ribosomes Role Signal Transduction Stress System Testing Tissues Translations Up-Regulation acute stress biological adaptation to stress gene conservation genetic approach glycosylation human disease human tissue in vivo in vivo Model in vivo imaging insight loss of function mRNA Translation mechanical properties mutant novel posttranscriptional protein complex response ribosome profiling sensor solute transcriptome sequencing whole genome

项目摘要

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.

项目概要细胞应激反应在细胞和生物生存中发挥着重要作用，并有助于人类广泛的生理过程和疾病的分子结构。大多数应激反应途径都有明确的定义，但渗透应激是一个显着的例外。反应，动物的相关压力传感器和信号机制很差大多数渗透应激反应的研究都使用体内培养的细胞。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，与人类一样，线虫通过新陈代谢来应对渗透压。葡萄糖产生有机渗透剂，例如甘油，我们进行了无偏向前研究。遗传筛选以鉴定不表现出渗透剂生物合成基因诱导的突变体（Nio 基因）并发现了 nio-2 的多个等位基因，它编码唯一的秀丽隐杆线虫同源物 O-GlcNAc 转移酶（ogt-1；人类 OGT）。胞质和核蛋白的 Ser/Thr 残基，还表现出重要的 GlcNAc 酰化缺乏 OGT 的哺乳动物细胞无法生存，但缺乏 OGT 的秀丽隐杆线虫 1 具有活力和繁殖能力，为研究 ogt-1 在细胞中的作用提供了独特的机会 ogt-1 突变体无法适应高渗环境并生长。有机渗透剂水平降低，并且不诱导渗透剂生物合成蛋白 GPDH-1。然而，渗透剂生物合成基因 mRNA 的渗透诱导是正常的，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弥补。或催化失活的人类 OGT，表明 OGT 在渗透压中的非典型功能应激反应从线虫到人类都是保守的。我们还发现了突变。保守的 3' mRNA 加工复合物的相互作用成分，可表型 ogt-1。证实ogt-1的非典型功能促进应激诱导的mRNA上调在渗透压过程中通过与 3’RNA 加工复合蛋白的相互作用进行翻译。检验这个假设，我们将确定时间、功能和监管要求 ogt-1 在渗透应激反应（目标 1）中的作用，确定特定的基因表达机制受 ogt-1 影响的细胞（目标 2），并确定 ogt-1 是否调节渗透应激反应通过与我们的 Nio 中也发现的 3' mRNA 切割和聚腺苷酸化成分的相互作用（目标 3）我们的研究将描绘压力信号传导的新范式并揭示新的模式。 OGT 影响细胞生理学的机制。