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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Research Article Metabolism

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 2

iCLIP-Seq identifies PSPC1 binding sites in adipocyte transcripts.

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iCLIP-Seq identifies PSPC1 binding sites in adipocyte transcripts. (A) I...
(A) Immunoblot analysis of PSPC1 and PPARγ in retrovirally derived 10T1/2 stable cell lines expressing WT PSPC1 or an RRM point mutant (RNP Mut). Quantification of PSPC1 bands normalized to HMG1 bands is indicated. (B) Real-time PCR analysis of gene expression in 10T1/2 stable cells described in A. Cells were stimulated to differentiate with DMI + 20 nM GW for 7 days. Comparison was made against vector control by 1-way ANOVA. Results represent 3 independent experiments. (C) Autoradiograph of cross-linked PSPC1-RNA complexes using denaturing gel electrophoresis. PSPC1-RNA complexes were immunopurified from 10T1/2 cells expressing vector or Flag-tagged PSPC1 (Fl-Pspc1) using Flag antibody. RNA was partially or fully digested using different concentrations of MNase. Complexes extending 20–40 kDa upward from the size of PSPC1 (59 kDa) were excised for further analysis (area indicated in squares). (D and E) PSPC1 iCLIP-Seq clusters mapped to the mouse EBF1 and ACSL1 gene (University of California, Santa Cruz, Genome Browser track). Four tracks in green, blue, yellow, and red represent day 0 vector, day 0 Fl-Pspc1, day 6 vector, and day 6 Fl-Pspc1, respectively. Peaks are piles of 30-nt significant clusters (cross-linking site ± 15 nt). Height of the peaks indicates the number of clusters identified within the region. Results are representative of 2 independent iCLIP-Seq experiments. Error bars represent mean + SEM. *P < 0.05, **P < 0.01.