Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice
Kenji Hata, … , Vincent R. Harley, Toshiyuki Yoneda
Kenji Hata, … , Vincent R. Harley, Toshiyuki Yoneda
Published August 1, 2008
Citation Information: J Clin Invest. 2008;118(9):3098-3108. https://doi.org/10.1172/JCI31373.
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Research Article Bone biology

Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

Abstract

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

Authors

Kenji Hata, Riko Nishimura, Shuji Muramatsu, Akio Matsuda, Takuma Matsubara, Katsuhiko Amano, Fumiyo Ikeda, Vincent R. Harley, Toshiyuki Yoneda

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Figure 2

Importance of association of p54nrb with Sox9 in upregulation of Col2a1 gene promoter activity.

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Physical and functional interactions of p54nrb with Sox9. (A) Stimul...
(A) Inhibition of p54nrb-stimulated Col2a1 promoter activity by dominant-negative Sox9 (DN). Luciferase activity of ATDC5 cell lysates transfected with Col2a1 luciferase construct, together with expression vectors as indicated was measured. (B) No association of dominant-negative Sox9 with p54nrb. The cell lysates expressing wild-type or the mutants of HA-Sox9 were precipitated (Ppt) with His-tag-p54nrb protein, and then the precipitates were determined by immunoblotting with anti-HA antibody. ΔHMG, a mutant lacking the HMG domain. (C) Schematic diagram of the mutants of p54nrb. ΔM, Δ224, and Δ313 are mutants of p54nrb. (D) Analysis of binding domain of p54nrb with Sox9. The cell lysates expressing wild-type or mutants of p54nrb were precipitated with tandem affinity purification–tagged (TAP-tagged) Sox9 protein, and then the precipitates were determined by immunoblotting with anti-Myc antibody. (E) The mutant p54nrb (ΔM) failed to transactivate the transcriptional activity of Sox9. ATDC5 cells were transfected with constructs as indicated, and luciferase activity of cell lysates was measured. (F) Knockdown of p54nrb by shRNA. shRNA expression vector for GFP or p54nrb (shGFP or shp54nrb) was transfected into ATDC5 cells, and the total RNA of the cells was determined by RT-PCR analyses. (G) Knockdown of p54nrb by shRNA. ATDC5 cells were transfected with shGFP or shp54nrb, and the cell lysates were examined by immunoblotting with anti-p54nrb and β-actin antibodies. (H) Inhibition of Sox9-dependent Col2a1 promoter activity by knockdown of p54nrb. Luciferase activity was measured in cell lysates transfected with expression vectors as indicated.