Post-transcriptional Regulation of PPAR-g Expression by 5'-Untranslated Regions

5-非翻译区对 PPAR-g 表达的转录后调控

• 批准号: 7880682
• 负责人: JHEEM D MEDH
• 金额: $ 15.68万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7880682
• 关键词: 5' Untranslated Regions; Affinity Chromatography; Amino Acids; Atherosclerosis; Binding; Binding Proteins; Biological; Biological Assay; California; Cells; Complementary DNA; Complex; Culture Media; Cytoplasm; Dactinomycin; Diabetes Mellitus; Digestion; Electrophoretic Mobility Shift Assay; Ensure; Enzyme-Linked Immunosorbent Assay; Eukaryotic Cell; Exons; Family; Gene Expression; Genes; Genetic Transcription; Half-Life; Human; In Vitro; Incubated; Individual; Label; Length; Ligands; Luciferases; Malignant Neoplasms; Measures; Messenger RNA; Monkeys; Northern Blotting; Nuclear Receptors; Obesity; Oligonucleotides; Open Reading Frames; Oryctolagus cuniculus; Outcome; Peptides; Physiologic pulse; Physiological; Post-Transcriptional Regulation; Process; Protein Binding; Protein Isoforms; Proteins; Public Health; RNA; RNA Probes; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Radioactivity; Radiolabeled; Regulation; Relative (related person); Reporter Genes; Reticulocytes; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease T1; Role; Secondary to; Sequence Analysis; Specificity; Stimulus; Structure; Subfamily lentivirinae; System; Techniques; Terminator Codon; Therapeutic; Time; Transcript; Translating; Translation Initiation; Translations; Undifferentiated; Universities; Untranslated Regions; Uridine; Variant; Virus; Western Blotting; base; cis acting element; crosslink; expression vector; human disease; in vivo; inhibitor/antagonist; innovation; mRNA Decay; mRNA Expression; mRNA Stability; mRNA capping; macrophage; member; migration; monocyte; novel; nuclease; polypeptide; protein expression; radiotracer; research study; response; stem; transcription factor; ultraviolet irradiation

PPAR-g is a member of the nuclear receptor family of transcription factors and is known to regulate many different genes with diverse physiological functions. The presence of a total of seven PPAR-g transcript isoforms has previously been demonstrated in monkey and human macrophages. Most of the variability between different PPAR-g transcripts is in the 5'-untranslated region (5'-UTR), such that the seven transcripts encode for only 3 different protein isoforms. Recently, 5'-UTRs have emerged as major modulators of cytoplasmic mRNA processing in eukaryotic cells. Such post-transcriptional regulation allows for rapid adjustments in protein expression in response to various stimuli. Based on experimental evidence, we hypothesize that sequence variations in the 5' UTR of PPAR-g transcripts may regulate mRNA stability ortranslational efficiency. The proposed studies focus on identifying mechanisms for the post-transcriptional regulation of PPARg expression by PPAR-g 5'-UTRs. The translation of different Lentivirus-derived PPAR-g transcript isoforms will be compared in THP-1 macrophages. A requirement for any macrophage-specific or ligand-induced factors will also be ascertained. Effect of PPAR-g 5'-UTR on translational efficiency will be investigated by in-vitro and in-vivo translation of full-length PPAR-g transcripts and of chimeric constructs of different PPARg 5'-UTR cloned upstream of the luciferase reporter gene. The stability (half-life) and decay rates of PPARg transcripts with different 5'-UTRs will be determined in the presence of transcription inhibitors by RT-PCR, Northern blot analysis and pulse-chase radiolabeling experiments. The presence of PPAR-g 5'-UTRspecific cytosolic RNA-binding proteins will be identified by electrophoretic mobility shift assays, UV crosslinking and affinity chromatography. The proposed studies will explain the biological significance of multiple PPAR-g transcripts and facilitate the use of PPAR-g 5'-UTR as targets for specific therapeutic outcomes. Relevance to Public Health: PPAR-g are implicated in many human diseases including atherosclerosis, diabetes, obesity and certain cancers. Information about posttranscriptional regulation of PPAR-g function is vital for efficient and selective modulation of different PPAR-g isoforms.

PPAR-g 是转录因子核受体家族的成员，已知可调节 许多不同的基因具有不同的生理功能。总共存在 7 个 PPAR-g 转录亚型先前已在猴子和人类巨噬细胞中得到证实。大部分的 不同 PPAR-g 转录本之间的变异性位于 5'-非翻译区 (5'-UTR)，因此 七个转录本仅编码 3 种不同的蛋白质亚型。 最近，5'-UTR 已成为真核生物细胞质 mRNA 加工的主要调节剂。 细胞。这种转录后调节允许快速调整蛋白质表达以响应 各种刺激。基于实验证据，我们假设 5' UTR 中的序列变异 PPAR-g 转录物可以调节 mRNA 稳定性或翻译效率。 拟议的研究重点是确定 PPARg 转录后调控机制 通过 PPAR-g 5'-UTR 表达。不同慢病毒衍生的 PPAR-g 转录亚型的翻译 将在 THP-1 巨噬细胞中进行比较。对任何巨噬细胞特异性或配体诱导的要求 因素也将被确定。 PPAR-g 5'-UTR 对翻译效率的影响将通过以下方式研究 全长 PPAR-g 转录物和不同 PPARg 嵌合构建体的体外和体内翻译 5'-UTR 克隆到荧光素酶报告基因的上游。 PPARg 的稳定性（半衰期）和衰减率 在存在转录抑制剂的情况下，通过 RT-PCR 确定具有不同 5'-UTR 的转录本， Northern 印迹分析和脉冲追踪放射性标记实验。 PPAR-g 5'-UTR 特异性的存在 胞质 RNA 结合蛋白将通过电泳迁移率变动分析、UV 交联来鉴定 和亲和层析。拟议的研究将解释多种的生物学意义 PPAR-g 转录并促进使用 PPAR-g 5'-UTR 作为特定治疗结果的靶点。 与公共卫生的相关性：PPAR-g 与许多人类疾病有关，包括 动脉粥样硬化、糖尿病、肥胖和某些癌症。有关转录后调控的信息 PPAR-g 功能对于有效、选择性地调节不同 PPAR-g 亚型至关重要。