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: 7131841
• 负责人: JHEEM D MEDH
• 金额: $ 11.63万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7131841
• 关键词: RNA binding protein; affinity chromatography; crosslink; gel mobility shift assay; genetic transcription; macrophage; messenger RNA; northern blottings; peroxisome proliferator activated receptor; polymerase chain reaction; protein isoforms; pulse radiolysis; receptor expression

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是转录因子的核受体家族的成员，已知会调节 许多具有不同生理功能的不同基因。总共存在七个PPAR-G 先前在猴子和人类巨噬细胞中证明了转录本同工型。大多数 不同PPAR-G转录本之间的可变性在5'-非翻译区域（5'-UTR）中，因此 七个转录本仅针对3种不同的蛋白质同工型进行编码。 最近，在真核生物中，有5'-UTR是细胞质mRNA加工的主要调节剂 细胞。这种转录后调节允许对蛋白质表达进行快速调整。 各种刺激。根据实验证据，我们假设5'UTR的序列变化 PPAR-G转录本可以调节mRNA稳定性或转移效率。 拟议的研究重点是识别PPARG转录后调控的机制 PPAR-G 5'-UTRS的表达。不同慢病毒衍生的PPAR-G转录本同工型的翻译 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要求 还将确定因素。 PPAR-G 5'-UTR对翻译效率的影响将由 全长PPAR-G转录本和不同PPARG的嵌合构建体的体外和体内翻译 5'-UTR克隆了荧光素酶报告基因的上游。 PPARG的稳定性（半衰期）和衰减率 具有不同5'-UTRS的转录本将在RT-PCR的转录抑制剂存在下确定 北印迹分析和脉搏射线标记实验。 PPAR-G 5'-utrspific的存在 胞质RNA结合蛋白将通过电泳迁移率分析，紫外线交联确定 和亲和力色谱。拟议的研究将解释多重的生物学意义 PPAR-G转录本，并促进使用PPAR-G 5'-UTR作为特定治疗结果的靶标。 与公共卫生相关：PPAR-G与许多人类疾病有关 动脉粥样硬化，糖尿病，肥胖和某些癌症。有关转录后调节的信息 PPAR-G功能对于对不同PPAR同工型的有效和选择性调节至关重要。