RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs
Jiexin Wang, … , Douglas L. Black, Peter Tontonoz
Jiexin Wang, … , Douglas L. Black, Peter Tontonoz
Published February 13, 2017
Citation Information: J Clin Invest. 2017;127(3):987-1004. https://doi.org/10.1172/JCI89484.
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RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs

Abstract

A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3′-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional control of adipose development and function.

Authors

Jiexin Wang, Prashant Rajbhandari, Andrey Damianov, Areum Han, Tamer Sallam, Hironori Waki, Claudio J. Villanueva, Stephen D. Lee, Ronni Nielsen, Susanne Mandrup, Karen Reue, Stephen G. Young, Julian Whitelegge, Enrique Saez, Douglas L. Black, Peter Tontonoz

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Figure 5

PSPC1 interacts with DDX3X and translocates from nucleus to cytoplasm during differentiation.

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PSPC1 interacts with DDX3X and translocates from nucleus to cytoplasm du...
(A) Silver staining of Flag-tagged PSPC1 (Fl-Pspc1) coimmunoprecipitated proteins on a gradient Bis-Tris gel. Co-IP with Flag antibody was performed in 10T1/2 cells stably expressing Fl-Pspc1 or vector. Arrows indicate bands present exclusively in the Fl-Pspc1 lane. (B) Immunoblot analysis of proteins coimmunoprecipitated with Fl-Pspc1 in 10T1/2 cells on differentiation day 0 and day 6. Cells were stimulated to differentiate with DMI + 20 nM GW. The known interaction partner NONO served as a positive control. Results are representative of 3 independent experiments. (C) Immunoblot analysis of DDX3X coimmunoprecipitated with Fl-Pspc1 in 10T1/2 cells on differentiation day 6 (middle panel). Cell lysates were treated with control or RNase prior to IP (right panel). Results are representative of 2 independent experiments. (D) Immunoblot analysis of DDX3X and NONO co-IP with PSPC1 and RRM mutants (ΔRRM1 and ΔRRM2). 10T1/2 stable cells were stimulated to differentiate with DMI + 20 nM GW. Results are representative of 2 independent experiments. (E) Immunoblot analysis of PSPC1 and NONO in the nuclear (Nuc) and cytoplasmic (Cyto) fractions of 10T1/2 cells on differentiation days 0 and 7. Cells were stimulated to differentiate with DMI + 20 nM GW. HMG1 and α-tubulin served as nuclear and cytoplasmic markers, respectively. Results are representative of 3 independent experiments. (F) PSPC1 subcellular localization visualized by fluorescent confocal microscopy in undifferentiated (Pre) and differentiated (Ad) 10T1/2 adipocytes stably expressing V5-tagged PSPC1 (Psp-V5). Cells were stimulated to differentiate with DMI + 20 nM GW for 7 days. Scale bars: 20 μm. Results are representative of 2 independent experiments. (G) PSPC1 subcellular localization visualized by fluorescent confocal microscopy in undifferentiated (Pre) and differentiated (Ad) 10T1/2 adipocytes stably expressing untagged PSPC1. Antibody against PSPC1 (red) was used for detecting endogenous PSPC1. Scale bars: 5 μm. Results are representative of 3 independent experiments.