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 1

Physical and functional interactions of p54nrb with Sox9.

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Colocalization of p54nrb with Sox9 in paraspeckle nuclear bodies. (A...
(A) Stimulation of Col2a1 promoter activity by cDNA clones isolated from ATDC5. ATDC5 cells were transfected with each cDNA together with the Col2a1-luciferase construct, and luciferase activity of cell lysates was measured. Clone 3 (cl 3) is p54nrb. Cont, control. (B) Costimulatory effect of p54nrb and Sox9 on Col2a1 promoter activity. ATDC5 cells were transfected with p54nrb together with Sox9, and luciferase activity of cell lysates was measured. (C) No upregulation of Col2a1 promoter activity by p54nrb in HeLa cells. HeLa cells were transfected with p54nrb together with Sox9, and luciferase activity of cell lysates was measured. (D) Absence of effect of p54nrb on transcriptional activity of Runx2. The osteocalcin gene promoter luciferase constructs were transfected into C3H10T1/2 cells with the plasmid as indicated. The luciferase activity of cell lysates was measured. (E) Physical association of p54nrb with Sox9. Cell lysates expressing Myc-p54nrb, HA-Sox9, or both were determined by immunoblotting with anti-HA antibody, followed by immunoprecipitation with anti-Myc antibody.