Volume 6, Issue 2, Pages 307-316 (August 2000) Direct Coupling of Transcription and mRNA Processing through the Thermogenic Coactivator PGC-1  María Monsalve, Zhidan Wu, Guillaume Adelmant, Pere Puigserver, Melina Fan, Bruce M. Spiegelman  Molecular Cell  Volume 6, Issue 2, Pages 307-316 (August 2000) DOI: 10.1016/S1097-2765(00)00031-9

Figure 1 PGC-1 Has Putative mRNA Processing Domains (A) Schematic representation of PGC-1 and mutant alleles. (B) Transcriptional activity of wild-type PCC-1 and C-terminal alleles on a UCP-2-luc reporter gene. Two micrograms of a UCP2-luc reporter plasmid, 0.3 μg of pSV- RXRα, 0.3 μg of pSV-PPARγ2, and 10 μg of pSV-PGC-1 were cotransfected into C2C12 cells, harvested 48 hr posttransfection, and the luciferase activity in the extracts was determined. (C) RNA analysis of PGC-1-expressing cells. C2C12 myoblasts expressing wild-type PGC-1 or the C-terminal alleles from retroviral pBABE vectors were allowed to reach confluency before total RNA was extracted and subjected to Northern blot analysis. Probes used for hybridization were PGC-1, NRF-1, NRF-2, mtTFA, UCP-2, and 36B4. The last is used as a control for equivalence of RNA quantities. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)

Figure 2 PGC-1 Colocalizes with Splicing Factors in Nuclear Speckles C2C12 cells were transfected with GFP-PGC-1 fusions and fixed 36 hr posttransfection. (A) Distribution of GFP-PGC-1 in the cell nucleus. (B) Colocalization of PGC-1 splicing factors was determined by immunofluorescence using antibodies against U1-70Kd. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)

Figure 3 The Phosphorylation State of the Cell Modulates the Nuclear Distribution of PGC-1 NIH3T3 cells were transfected with GFP-PGC-1 and 24 hr posttransfection were treated for 1 hr with vehicle, 50 μM staurosporine, or 1 μM okadaic acid, fixed with 3.7% formaldehyde for 10 min, and mounted for microscopic observation. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)

Figure 4 PGC-1 Associates with Splicing Factors through Its C-Terminal Domain 293T cells were transfected, and 24 hr posttransfection whole-cell extracts were prepared for IP. (A) Flag-PGC-1 (F-PGC-1) and Flag-PGC-1-ΔCTD (F-ΔCTD) were immunoprecipitated, and the precipitates were then subjected to PAGE and probed with α-SR40 (which cross-reacts with several other SR proteins) by Western blotting. Transiently transfected 293T cells expressed the Flag-tagged version of PGC-1 about 3× above the endogenous PGC-1 levels. (B) Experiments were performed as in (A), except that α-SC-35 was used to IP prior to Western blotting for PGC-1. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)

Figure 5 PGC-1 Associates with the Elongating Form of Pol II Flag-PGC-1 and Flag-PGC-1-ΔCTD were immunoprecipitated, and precipitates were probed with antibodies against Pol II (c-21, H5), nucleolin, CdPSF100Kd, cdk9, and cyclin T by Western blotting. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)

Figure 6 PGC-1 Regulation of mRNA Processing In Vivo (A) 293T cells were transfected with 3 μg of the FN minigene 7iBi89 and 3 μg of pCMV-HRS or the 3.1-Flag-PGC-1 vector. (B) 293T cells were transfected with 3 μg of pDR-1/FN, and were indicated, 0.6 μg of pSV-RXRα, 0.6 μg of pSV-PPARγ2, 3 μg of 3.1-Flag-PGC-1, 3 μg of 3.1-Flag-PGC-1-ΔCTD, 3 μg of pCMV-HRS. (C) Experiments were performed as in (B), except that 120 ng of pDR-1/FN was used. Total RNA was extracted 24 hr posttransfection and subjected to RT–PCR analysis. The identity of the −IIB and +IIIB PCR products was confirmed by DNA sequencing. The intermediate band in (C) (*) is of unknown origin. Loading control PCR reactions that amplified part of the endogenous FN transcript were carried out in all cases. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)

Figure 7 Schematic Representation of PGC-1 Action (A) PGC-1 is loaded by specific transcription factors and associates with the PIC. (B) As transcription initiates, the CTD of Pol II is phosphorylated, and a set of proteins associates with it, including PGC-1 through its C-terminal region. (C) The elongation complex that carries PGC-1 regulates the coordinated regulation of mRNA processing. TBP, TATA-binding protein; HAT, histone acetyl transferase. Molecular Cell 2000 6, 307-316DOI: (10.1016/S1097-2765(00)00031-9)