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 5

Impairment of paraspeckle formation and Col2a1 mRNA processing by a mutant p54nrb (Δ244).

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Impairment of paraspeckle formation and Col2a1 mRNA processing by a muta...
(A) Impairment of paraspeckle body formation by a p54nrb mutant. ATDC5 cells transfected with Venus-tagged wild-type or mutant p54nrb and DsRed-PSP1 were visualized under a fluorescence microscope. Nucleuses of cells were stained with DAPI. (B) Effect of the mutant p54nrb on proliferation of ATDC5 cells. Control, wild-type p54nrb, or the mutant p54nrb were transfected into ATDC5 cells, and the growth of the cells was determined by cell proliferation assay. (C) Wild-type and a mutant p54nrb does not affect localization of SC-35. ATDC5 cells transfected with Venus-tagged wild-type or mutant p54nrb were immunostained with anti–SC-35 antibody. (D) Upregulation of transcriptional activity of Sox9 by a p54nrb mutant. ATDC5 cells were transfected with wild-type and a mutant p54nrb together with Sox9, and luciferase activity of cell lysates was measured. (E) Colocalization of a mutant p54nrb with Sox9. ATDC5 cells transfected with DsRed-tagged Sox9 and Venus-tagged p54nrb mutant were monitored under a fluorescence microscope. (F) Impairment of Col2a1 mRNA processing by a mutant p54nrb. A minigene construct of the Col2a1 gene was transfected with expression plasmids as indicated into ATDC5 cells. Total RNA isolated from the cells was determined by RT-PCR analyses using the primers (see Methods) specific for the minigene products (upper panel) or β-actin (bottom panel). Original magnification, ×400 (A, C, and E).