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: 7648140
• 负责人: JHEEM D MEDH
• 金额: $ 16.18万
• 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/7648140
• 关键词: 5' Untranslated Regions; Affinity Chromatography; Amino Acids; Atherosclerosis; Binding; Binding Proteins; Biological; Biological Assay; 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; Pulse taking; RNA; RNA Probes; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Radioactivity; Radiolabeled; Rate; Regulation; Relative (related person); Reporter Genes; Reticulocytes; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease T1; Role; Secondary to; Sequence Analysis; Specificity; Standards of Weights and Measures; Stimulus; Structure; Subfamily lentivirinae; System; Techniques; Terminator Codon; Therapeutic; Time; Transcript; Translating; Translation Initiation; Translations; Undifferentiated; 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; size; 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，并且已知其调节转录因子的表达。 许多具有不同生理功能的不同基因。共存在7个PPAR-g 先前已在猴和人巨噬细胞中证实了转录物同种型。大部分 不同PPAR-g转录物之间的可变性在5 '-非翻译区（5'-UTR）中，使得PPAR-g转录物的可变性在5 '-非翻译区（5'-UTR）中。 7个转录物仅编码3种不同的蛋白质同种型。 最近，5 '-UTR已成为真核细胞胞质mRNA加工的主要调节剂 细胞这种转录后调节允许响应于蛋白质表达的快速调节。 各种刺激。基于实验证据，我们假设， PPAR-g转录物可调节mRNA的稳定性或翻译效率。 这些研究的重点是确定PPARg的转录后调控机制 通过PPAR-g 5 '-UTR表达。不同慢病毒衍生的PPAR-g转录异构体的翻译 将在THP-1巨噬细胞中进行比较。任何巨噬细胞特异性或配体诱导的 因素也将得到确认。PPAR-g 5 '-UTR对翻译效率的影响将通过以下方法研究： 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克隆在荧光素酶报告基因的上游。PPARg的稳定性（半衰期）和衰变率 在转录抑制剂存在下通过RT-PCR测定具有不同5 ′-UTR的转录物， 北方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 细胞溶质RNA结合蛋白将通过电泳迁移率变动分析、UV交联 和亲和层析。拟议的研究将解释多种生物学意义， PPAR-g转录物，并促进PPAR-g 5 '-UTR作为特定治疗结果的靶标的使用。 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、肥胖症和某些癌症。转录后调控的信息 PPAR-g功能对于不同PPAR-g亚型的有效和选择性调节至关重要。