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.
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