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

PPAR-g是核受体转录因子家族中的一员，已知能调节 许多不同的基因具有不同的生理功能。共存在7个PPAR-g 此前已经在猴子和人类巨噬细胞中发现了转录异构体。大多数 不同PPAR-g转录本之间的可变性在5‘-非翻译区(5’-UTR)，因此 7个转录本只编码3种不同的蛋白质亚型。 近年来，5‘-UTRs已成为真核生物细胞质中mRNA加工的主要调节因子 细胞。这种转录后调控允许蛋白质表达的快速调整，以响应 各种刺激。根据实验证据，我们推测，在5‘非编码区的序列变异 PPAR-g转录本可能调节mRNA的稳定性或翻译效率。 建议的研究集中在确定PPARg转录后调控的机制 PPAR-g 5‘-UTRs的表达。慢病毒来源的不同PPAR-g转录体的翻译 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 PPAR-g全长转录本和不同PPAR-g嵌合结构的体外和体内翻译 5‘端非编码区克隆在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的转录本将在存在转录抑制物的情况下通过RT-PCR进行检测， Northern印迹分析和脉冲追逐标记实验。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改变分析、UV交联 亲和层析。拟议的研究将解释多重基因的生物学意义。 PPAR-g转录本，并促进使用PPAR-g 5‘-UTR作为特定治疗结果的靶点。 与公共卫生相关：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。关于转录后调控的信息 PPAR-g功能对于有效和选择性地调节不同的PPAR-g亚型至关重要。

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.