Paraspeckle protein p54^nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

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 (p54^nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54^nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54^nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54^nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54^nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54^nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54^nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54^nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54^nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

Chondrogenesis is an important biological event in endochondral bone development, skeletogenesis, and tissue patterning (1, 2). After condensation of chondrogenic mesenchymal cells, they begin to differentiate into chondrocytes (3). The transcription factor Sox9 contains a SRY-related high-mobility group box and promotes chondrocyte differentiation and the expression of cartilage-specific extracellular matrix genes, including collagens and proteoglycans (3). In humans, heterozygous SOX9 mutations cause campomelic dysplasia characterized by severe chondrodysplasia (4, 5). Heterozygous Sox9 mutant mice or mice lacking Sox9 function show impaired endochondral bone formation (6, 7). These findings indicate that Sox9 plays an essential role in chondrogenesis.

Sox9 is also implicated in the expression of Sox5 and Sox6, both of which form a transcriptional complex with Sox9 to control the expression of collagen, type II, α 1 (Col2a1) and aggrecan (Acan) during chondrocyte differentiation (7). In addition, PGC-1α functions as a transcriptional coactivator for Sox9 (8). Moreover, the functional and physical interaction of Sox9 and β-catenin is important evidence for crosstalk between Sox9 and Wnt signaling (9). These findings have contributed to identification of transcriptional components for Sox9 that are implicated in chondrogenesis. However, other modes of the regulation of chondrogenic genes, including chromatin remodeling, histone modification, and RNA processing, are not well understood.

Because the transcription factors form a large complex that sequentially and dynamically controls expression of target genes in collaboration with the partner molecules, identification of the components of the “transcriptional factory” formed by Sox9 would lead to a breakthrough in our understanding of the mechanisms of Sox9-regulated chondrogenesis. In this study, we identify 54-kDa nuclear RNA-binding protein (p54^nrb) as an interacting partner of Sox9 and show that p54^nrb controls transcription and mRNA processing of the Col2a1 gene. Furthermore, we demonstrate that p54^nrb plays an important role in chondrogenesis in vitro and in vivo. Thus p54^nrb is an essential transcriptional regulator that links the Sox9-dependent transcription to target gene mRNA maturation during chondrogenesis.

Physical and functional interaction of p54^nrb with Sox9. To identify factors involved in Sox9-mediated regulation of chondrocyte differentiation, we screened a cDNA library generated from the chondrogenic cell line, ATDC5 (10), by performing a luciferase reporter assay using the Col2a1 gene promoter. Four cDNA clones stimulated the Col2a1 gene promoter (Figure 1A). One of these encodes the full-length p54^nrb protein (11–13), which contains 2 RNA recognition motifs (RRMs) and is localized to nuclear paraspeckle bodies (14). We have confirmed that p54^nrb mRNA is expressed in ATDC5 cells and in primary mouse chondrocytes (data not shown). To examine the functional relationship between p54^nrb and Sox9, we determined the effect of p54^nrb on the transcriptional activity of Sox9. Overexpression of p54^nrb markedly enhanced Sox9 transactivation of the Col2a1 gene promoter (Figure 1B). We did not observe the upregulation of the Col2a1 promoter activity by p54^nrb in HeLa cells where Sox9 is not expressed (Figure 1C). In contrast, p54^nrb did not affect the transcriptional activity of Runx2, an essential transcription factor for osteoblast differentiation (15) (Figure 1D), suggesting that p54^nrb specifically stimulates the transcriptional activity of Sox9. To investigate the basis of the functional cooperation between p54^nrb and Sox9, we tested whether p54^nrb and Sox9 proteins interact. As shown in Figure 1E, coimmunoprecipitation experiments indicated a physical association between p54^nrb and Sox9. These results indicate that p54^nrb functions as a transcriptional partner for Sox9. Next we attempted to define whether the functional interaction between p54^nrb and Sox9 is required for regulation of the Col2a1 gene promoter activity. Overexpression of a dominant-negative Sox9 mutant, which lacks binding activity to p54^nrb, markedly inhibited stimulation of Col2a1 promoter activity by p54^nrb (Figure 2, A and B). A mutant of p54^nrb, ΔM, which lacks binding activity to Sox9 (Figure 2, C and D), failed to stimulate the transcriptional activity of Sox9 (Figure 2E). Furthermore, knockdown of p54^nrb (Figure 2, F and G) clearly inhibited transcriptional activity of Sox9 on the Col2a1 gene promoter (Figure 2H). These results demonstrated that p54^nrb is a critical transcriptional partner of Sox9 and that this partnership upregulates Col2a1 gene promoter activity.

Figure 1

Physical and functional interactions of p54^nrb with Sox9. (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 p54^nrb. Cont, control. (B) Costimulatory effect of p54^nrb and Sox9 on Col2a1 promoter activity. ATDC5 cells were transfected with p54^nrb together with Sox9, and luciferase activity of cell lysates was measured. (C) No upregulation of Col2a1 promoter activity by p54^nrb in HeLa cells. HeLa cells were transfected with p54^nrb together with Sox9, and luciferase activity of cell lysates was measured. (D) Absence of effect of p54^nrb 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 p54^nrb with Sox9. Cell lysates expressing Myc-p54^nrb, HA-Sox9, or both were determined by immunoblotting with anti-HA antibody, followed by immunoprecipitation with anti-Myc antibody.

Figure 2

Importance of association of p54^nrb with Sox9 in upregulation of Col2a1 gene promoter activity. (A) Inhibition of p54^nrb-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 p54^nrb. The cell lysates expressing wild-type or the mutants of HA-Sox9 were precipitated (Ppt) with His-tag-p54^nrb 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 p54^nrb. ΔM, Δ224, and Δ313 are mutants of p54^nrb. (D) Analysis of binding domain of p54^nrb with Sox9. The cell lysates expressing wild-type or mutants of p54^nrb 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 p54^nrb (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 p54^nrb by shRNA. shRNA expression vector for GFP or p54^nrb (shGFP or shp54^nrb) was transfected into ATDC5 cells, and the total RNA of the cells was determined by RT-PCR analyses. (G) Knockdown of p54^nrb by shRNA. ATDC5 cells were transfected with shGFP or shp54^nrb, and the cell lysates were examined by immunoblotting with anti-p54^nrb and β-actin antibodies. (H) Inhibition of Sox9-dependent Col2a1 promoter activity by knockdown of p54^nrb. Luciferase activity was measured in cell lysates transfected with expression vectors as indicated.

Interaction of p54^nrb and Sox9 in paraspeckle nuclear bodies. It has been reported that p54^nrb colocalizes and interacts with paraspeckle protein 1 (PSP1) in the paraspeckle nuclear bodies (14, 16). Although nuclear paraspeckles are assumed to be involved in nuclear events, including DNA replication, transcription, and mRNA maturation (17), their biological roles remain unclear. First, we determined the subcellular localization of p54^nrb in ATDC5 cells using Venus-tagged p54^nrb. p54^nrb was confined to the nucleus within nuclear speckles (Figure 3A) and colocalized with PSP1 (Figure 3A) but not with SC-35 (Figure 3B), a nuclear speckle body protein implicated in RNA splicing (18, 19). Consistent with results shown in Figure 1, p54^nrb is colocalized with Sox9 in transfected cells and primary mouse chondrocytes (Figure 3, C–F). Like p54^nrb, Sox9 colocalized with PSP1 but not with SC-35 (Figure 3, G and H). When Sox9 is co-overexpressed, the size of paraspeckle bodies became smaller (Figure 3, C and D). Because the size of nuclear speckle has been shown previously to be inversely correlated with the state of transcription of the cell (17), our data suggest that interaction between p54^nrb and Sox9 stimulates transcriptional activity.

Figure 3

Colocalization of p54^nrb with Sox9 in paraspeckle nuclear bodies. (A) Colocalization of p54^nrb with PSP1 in the nucleus of ATDC5 cells. ATDC5 cells transfected with Venus-tagged p54^nrb (Venus-p54^nrb) and DsRed-tagged PSP1 (DsRed-PSP1) were monitored under a fluorescence microscope. (B) p54^nrb is not localized with SC-35. ATDC5 cells transfected with Venus-tagged p54^nrb were immunostained with anti–SC-35 antibody. (C) Colocalization of p54^nrb and Sox9 in the nucleus. ATDC5 cells transfected with Venus-tagged p54^nrb and DsRed-tagged Sox9 (DsRed-Sox9) were monitored under a fluorescence microscope. (D and E) Colocalization of p54^nrb and Sox9 in the nucleus. C3H10T1/2 cells transfected with Venus-tagged p54^nrb and DsRed-tagged Sox9 were monitored under a confocal microscope. C3H10T1/2 cells expressing both Venus-p54^nrb and DsRed-Sox9 show smaller size of speckle than C3H10T1/2 cells expressing only Venus-p54^nrb (D). Nucleuses of cells were stained with DAPI. (F) Colocalization of p54^nrb and Sox9 in murine primary chondrocytes. Primary chondrocytes isolated from mouse ribs were immunostained with anti-p54^nrb and anti-Sox9 antibodies and photographed under a confocal microscope. (G) Colocalization of Sox9 with PSP1 in the nucleus of ATDC5 cells. ATDC5 cells transfected with DsRed-tagged Sox9 and Venus-tagged PSP1 (Venus-PSP1) were monitored under a fluorescence microscope. (H) Sox9 does not affect localization of SC-35. ATDC5 cells transfected with DsRed-tagged Sox9 were immunostained with anti-SC-35 antibody. Original magnification, ×400.

Role of p54^nrb in mRNA processing of Col2a1. Because 2 RRMs of p54^nrb are highly homologous to the RRM domain of poly-pyrimidine tract-binding protein–associated splicing factor (PSF), we speculated that p54^nrb is involved in splicing of the target genes necessary for chondrogenesis. We examined the processing of Col2a1 mRNA by performing a minigene assay using a Col2a1 gene cassette, containing the alternatively spliced exon 2 driven by the native promoter (20). Overexpression of Sox9 or p54^nrb produced a spliced transcript of the Col2a1 (IIA) containing exon 2 (20, 21) (Figure 4, A and B). Furthermore, co-overexpression of Sox9 and p54^nrb produced more transcripts of Col2a1 (IIA) than expression of Sox9 or p54^nrb alone (Figure 4, A and B). In addition, co-overexpression of Sox9 and p54^nrb also produced type II B isoform (Figure 4A), which is preferentially expressed in differentiated chondrocytes (21). However, p54^nrb is not involved in splicing of the fibronectin gene (Figure 4C). These results suggest that p54^nrb specifically controls the splicing and maturation of Col2a1 mRNA. To further understand the role of p54^nrb in this process, we generated a mutant of p54^nrb (Δ224), lacking 2 RRMs (Figure 2C). In ATDC5 cells in which the mutant was expressed, the shape of paraspeckle bodies was rounded (Figure 5A), without affecting the proliferation of ATDC5 cells (Figure 5B), and not colocalized with SC-35 (Figure 5C). Interestingly, however, the mutant p54^nrb still upregulated the transcriptional activity of Sox9 (Figure 5D) and retained the binding capacity to Sox9 (Figure 2D). In addition, where mutant p54^nrb colocalized with Sox9, the size of speckles was smaller (Figure 5E). Next, we evaluated the role of both the p54^nrb RRM and paraspeckle nuclear bodies in the processing of Col2a1 mRNA. Overexpression of the mutant p54^nrb blocked the maturation of Col2a1 mRNA (Figure 5F). We confirmed that this effect of the mutant p54^nrb is not due to inhibition of transcription of the minigene (Figure 4B). Collectively, the results indicate that p54^nrb regulates both the splicing of Col2a1 mRNA and the formation of paraspeckle bodies via RRMs.

Figure 4

Processing of Col2a1 mRNA by p54^nrb. (A) Col2a1 mRNA processing by p54^nrb. 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) Ex1, exon 1. (B) Effect of wild-type and a mutant p54^nrb on processing of Col2a1 mRNA. A minigene construct of the Col2a1 gene was transfected with the expression vectors as indicated into ATDC5 cells. Expression of spliced minigene product (upper panel) and nonspliced minigene product (bottom panel) was determined by real-time PCR using the primers (see Methods) specific for the spliced and nonspliced minigene products. Expression levels were normalized with β-actin expression. (C) Effect of p54^nrb on processing of fibronectin mRNA. HEK293 cells were transfected with fibronectin minigene construct (7iBi) together with mock (Cont), SRP40 expression vector, or p54^nrb expression vector. 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 GAPDH (bottom panel).

Figure 5

Impairment of paraspeckle formation and Col2a1 mRNA processing by a mutant p54^nrb (Δ244). (A) Impairment of paraspeckle body formation by a p54^nrb mutant. ATDC5 cells transfected with Venus-tagged wild-type or mutant p54^nrb and DsRed-PSP1 were visualized under a fluorescence microscope. Nucleuses of cells were stained with DAPI. (B) Effect of the mutant p54^nrb on proliferation of ATDC5 cells. Control, wild-type p54^nrb, or the mutant p54^nrb were transfected into ATDC5 cells, and the growth of the cells was determined by cell proliferation assay. (C) Wild-type and a mutant p54^nrb does not affect localization of SC-35. ATDC5 cells transfected with Venus-tagged wild-type or mutant p54^nrb were immunostained with anti–SC-35 antibody. (D) Upregulation of transcriptional activity of Sox9 by a p54^nrb mutant. ATDC5 cells were transfected with wild-type and a mutant p54^nrb together with Sox9, and luciferase activity of cell lysates was measured. (E) Colocalization of a mutant p54^nrb with Sox9. ATDC5 cells transfected with DsRed-tagged Sox9 and Venus-tagged p54^nrb mutant were monitored under a fluorescence microscope. (F) Impairment of Col2a1 mRNA processing by a mutant p54^nrb. 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).

Regulation of chondrocyte differentiation by p54^nrb. To investigate whether p54^nrb is involved in chondrogenesis, we examined its effect on chondrocyte differentiation. Overexpression of p54^nrb in ATDC5 cells induced the endogenous expression of Col2a1 and Acan (Figure 6, A–D). In contrast, knockdown of p54^nrb clearly inhibited Sox9-induced Col2a1 expression (Figure 6E). Next, we examined the effect of the p54^nrb mutant that impaired Col2a1 mRNA processing and paraspeckle body formation. Overexpression of the p54^nrb mutant dramatically inhibited Sox9-induced Col2a1 expression (Figure 6, C and D). Also, the mutant p54^nrb abolished bone morphogenetic protein 2–induced (BMP2-induced) chondrocyte differentiation (Figure 6F). On the other hand, the mutant p54^nrb did not affect BMP2-induced osteoblast differentiation (Figure 6G). Furthermore, organ culture experiments using mouse fetal metatarsals indicated that while wild-type p54^nrb stimulated chondrogenesis, the p54^nrb mutant suppressed it (Figure 6, H–J). These data indicate that p54^nrb plays a critical role in chondrocyte differentiation via its RRM domains.

Figure 6

Role of p54^nrb in the regulation of chondrocyte differentiation. (A) The p54^nrb-induced Col2a1 and Acan in ATDC5 cells. ATDC5 cells were transfected as indicated, and then subjected to RT-PCR analyses. (B) The p54^nrb-induced type IIA and IIB forms of Col2a1 in ATDC5 cells. ATDC5 cells were transfected as indicated, and then subjected to RT-PCR analyses using the specific primers (see Methods) for Col2a1 (upper panel; the primer set can distinguish 2 isoforms of Col2a1), Col2a1 (middle panel; the primer set cannot distinguish 2 isoforms of Col2a1), or β-actin. (C and D) ATDC5 cells were infected with Sox9 and/or Myc-tagged wild-type or a Myc-tagged mutant p54^nrb (Δ224) adenovirus and then subjected to northern (C) or western (D) blotting, respectively. (E) Knockdown of p54^nrb suppressed Sox9-induced Col2a1 expression. ATDC5 cells were transfected with expression vectors as indicated, and then total RNA of the cells was determined by RT-PCR analyses. (F) The p54^nrb mutant blocked chondrocyte differentiation. C3H10T1/2 cells were infected with Myc-tagged wild-type or mutant p54^nrb (Δ224) adenovirus in the presence or absence of BMP2. The cells were examined by alcian blue staining. (G) No effects of p54^nrb on osteoblastogenesis. C2C12 cells infected with Myc-tagged wild-type or mutant p54^nrb (Δ224) adenovirus in the presence or absence of BMP2. The cells were examined by alkaline phosphatase staining. (H and I) Requirement of p54^nrb for chondrogenesis. Metatarsals isolated from mouse embryo were infected with control, Myc-tagged wild-type p54^nrb, or a Flag-tagged mutant p54^nrb (Δ224) adenovirus, incubated for 6 days, and subjected to immunostaining (H), histological analysis (I), and statistically analyses of the samples (J). PZ, proliferating zone; HZ, hypertrophic zone. All data were analyzed by ANOVA, followed by Student’s t test. Values shown are mean ± SD. (*P < 0.05, **P < 0.01 versus control). Original magnification, ×50 (G); ×40 (I).

Requirement of p54^nrb in chondrogenesis in vivo. To demonstrate the importance of p54^nrb in chondrogenesis in vivo, we generated transgenic mice, in which the mutant p54^nrb is specifically expressed in the chondrocyte lineage, using the Col2a1 gene promoter (Figure 7, A–C). Transgenic mice showed dwarfism between the neonatal period and 3 weeks after birth (Figure 7, D–H). Histological analyses showed that the layer of proliferating chondrocytes was markedly thinner in the transgenic mice compared with wild-type littermates (Figure 7I). Consistent with the reduction in Col2a1 expression by overexpression of the mutant p54^nrb (Figure 6, C and D), Col2 expression was suppressed in the transgenic mice (Figure 7, J and K). These results indicate that p54^nrb plays a critical role in chondrocyte differentiation. To further understand the role of p54^nrb in chondrogenesis, we examined the transgenic mice at younger stage. As shown in Figure 8A, enchondral ossification at the birth was markedly impaired in the transgenic mice. In situ hybridization experiments also indicated the delayed chondrogenesis in the transgenic mice at E15 (Figure 8B). In contrast, consistent with the results shown in Figure 5B, proliferation of chondrocytes was not influenced in the transgenic mice. Collectively, these data suggest that inhibition of mRNA processing of Col2a1 by p54^nrb leads to delay of ossification, which resulted in dwarfism seen in the transgenic mice.

Figure 7

Impaired chondrogenesis in the transgenic mice expressing the p54^nrb mutant. (A) The expression of the transgene in the chondrocytes isolated from the transgenic mice. Total RNA of chondrocytes isolated from wild-type or the transgenic mice was determined by RT-PCR analysis using specific primer (see Methods) for the transgene (Δ224) (upper panel) or β-actin (bottom panel). (B) Specific expression of p54^nrb mutant in chondrocytes of the transgenic mice. Total RNA isolated from brain (lane 1), kidney (lane 2), heart (lane 3), liver (lane 4), spleen (lane 5), chondrocytes (lane 6), or osteoblasts (lane 7) of the transgenic mice was determined by RT-PCR using specific primer (see Methods) for transgene (Δ224) (upper panel) or β-actin (bottom panel). (C) Overexpression of the mutant p54^nrb in the transgenic mice. Total RNA of chondrocytes isolated from wild-type or the transgenic mice was determined by real-time PCR analysis using a Taqman probe, which recognizes both intact and the mutant p54^nrb. (D and E) Dwarfism of the transgenic mice at birth. (F and G) Dwarfism of the transgenic mice at 3 weeks. D and F are macroscopic photographs and E and G are x-rays of the mice. (H) Body size of the transgenic mice. (I) H&E-stained section of the tibias of wild-type and transgenic mice at 2 weeks. (J) Col2 expression in the tibias of wild-type and transgenic mice. (K) Reduction in Col2α1 expression in the transgenic mice. Total RNA of chondrocytes isolated from wild-type or the transgenic mice was determined by real-time PCR analysis using a Taqman probe for Col2a1. Original magnification, ×40 (I and J).

Figure 8

Delayed enchondral ossification in the transgenic mice. (A) The sections of wild-type and the transgenic mice at neonatal period were subjected to H&E, alcian blue, or von Kossa staining. (B) The sections of wild-type and the transgenic mice at E15 were determined by in situ hybridization (Col2a1, Col10a1, PTHR1, Ihh), H&E staining, and von Kossa staining. (C) Normal proliferation of chondrocytes in wild-type and the transgenic mice at E15. The sections of wild-type and the transgenic mice at E15 were determined by immunostaining with anti-PCNA antibody. Original magnification, ×25 (A); ×40 (B); ×20 (C, left panel of WT and Tg); ×40 (C, right panel of WT and Tg).

The expression of the genes necessary for chondrogenesis is exquisitely and harmoniously regulated by several transcription factors (1). Several lines of evidence demonstrate the indispensable role of Sox9 for the commitment of mesenchymal cells into chondrocytes and the early stages of chondrocyte differentiation (3). In the present study, we have identified p54^nrb as an important transcriptional partner of Sox9 during chondrogenesis. We found that p54^nrb stimulates the transcriptional activity of Sox9 and chondrogenic action of Sox9. In contrast, knockdown of p54^nrb inhibited the activity of Sox9. Consistently, a dominant-negative mutant of Sox9 dramatically suppressed the stimulatory effect of p54^nrb on Col2a1 promoter activity. The interaction between p54^nrb and Sox9 was confirmed by coimmunoprecipitation experiments and colocalization in the nucleus of ATDC5 cells and primary chondrocytes. Collectively, these results clearly indicate that p54^nrb forms a transcriptional factory with Sox9.

Because p54^nrb has been shown to play a role in regulation of splicing or termination of mRNA synthesis (22), we examined whether p54^nrb regulates the processing of Col2a1 mRNA. Minigene assay showed that p54^nrb overexpression stimulates the processing of Col2a1 mRNA. Moreover, the mutant of p54^nrb lacking RRMs inhibited Col2a1 mRNA processing. Consistently, the mutant p54^nrb also blocked Col2a1 expression induced by Sox9. However, interestingly, the mutant p54^nrb retains the ability to upregulate the transcriptional activity of Sox9 on the Col2a1 gene promoter. These results indicate that p54^nrb regulates both transcription and splicing of Col2a1 mRNA through 2 different functional domains during chondrocyte differentiation and that p54^nrb is a critical Sox9 transcriptional factory component that couples the transcription and processing of Col2a1 mRNA.

Several studies demonstrate that nuclear speckles are implicated in the regulation of transcription and splicing of target genes and, consequently, controlling of cell growth and differentiation (14, 23, 24). p54^nrb is also implicated in formation of paraspeckle bodies with PSP1 (14). However, the biological role of p54^nrb and paraspeckle body is unknown. In this study, we demonstrate that p54^nrb is critical for chondrogenesis. Overexpression of p54^nrb upregulated the expression of chondrogenic genes and stimulated chondrocyte differentiation in C3H10T1/2 cells. In the cells, knockdown experiments supported the importance of p54^nrb in chondrogenesis. In organ culture systems, p54^nrb stimulated chondrogenesis. Furthermore, overexpression of the p54^nrb mutant lacking RRMs suppressed chondrogenesis in vitro and in vivo. This mutation caused enlargement of paraspeckle size, without affecting the size of the authentic nuclear speckle formed by SC-35, which is also implicated in regulation of splicing of mRNA (18). Interestingly, Sox9 is localized in the paraspeckle body and decreased its size. Collectively, our results raise a possibility that the RRM domain interaction of p54^nrb with Sox9 dynamically regulates the paraspeckle formation and mRNA processing necessary for chondrocyte differentiation.

In conclusion, our findings provide what we believe to be the novel paradigm whereby linkage between transcription and mRNA processing by a paraspeckle regulatory protein is critical for chondrocyte development. Thus, this finding provides a novel insight to our knowledge into the molecular basis of Sox9 regulation and chondrogenesis.

We thank Benoit de Crombrugghe for the Col2a1 gene promoter, Angus Lamond for PSP1 cDNA, Gobinda Sarkar for Col2a1 minigene cassette DNA, and Atsushi Miyawaki for Venus construct. We thank Masahiro Saito and Eisaku Nishida for technical help for in situ hybridization. This work was supported in part by the Ministry of Education, Science, Sports and Culture Grant-in-Aid (to R. Nishimura and T. Yoneda), Grant-in-Aid for Scientific Research on Priority Areas B (to T. Yoneda and R. Nishimura), The 21st Century COE Program (to T. Yoneda and R. Nishimura), Kato Memorial Bioscience Foundation (to K. Hata), and the NHMRC Australia (to V.R. Harley).

Address correspondence to: Riko Nishimura, Department of Molecular and Cellular Biochemistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan. Phone: 81-6-6879-2887; Fax: 81-6-6879-2890; E-mail: rikonisi@dent.osaka-u.ac.jp.

Nonstandard abbreviations used: BMP, bone morphogenetic protein; Col2a1, collagen, type II, α 1; p54^nrb, 54-kDa nuclear RNA-binding protein; PSP1, paraspeckle protein 1; RRM, RNA recognition motif.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:3098–3108 (2008). doi:10.1172/JCI31373

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