TASR-1 regulates alternative splicing of collagen genes in chondrogenic cells

Abstract

During the differentiation of chondroprogenitors into mature chondrocytes, the alternative splicing of collagen genes switches from longer isoforms to shorter ones. To investigate the underlying mechanisms, we infected mouse ATDC5 chondroprogenitor cells with retrovirus for stable expression of two closely related SR splicing factors. RT-PCR analysis revealed that TASR-1, but not TASR-2, influenced alternative splicing of type II and type XI collagens in ATDC5 cells. The effect of TASR-1 on splicing could be reversed with the addition of insulin. Results from our microarray analysis of ATDC5 cells showed that TASR-1 and TASR-2 differentially affect genes involved in the differentiation of chondrocytes. Of special interest is the finding that TASR-1 could down-regulate expression of type X collagen, a hallmark of hypertrophic chondrocytes. Immunohistostaining demonstrated that TASR-1 protein is more abundantly expressed than TASR-2 in mouse articular chondrocytes, raising the possibility that TASR-1 might be involved in phenotype maintenance of articular chondrocytes.

Fig. 1. Retroviral expression of T7-tagged TASR proteins in ATDC5 cells

(A) The RNP consensus sequences shared by TASR-1 and TASR-2 are shown in gray boxes. RS domains are in hatched boxes. (B) Nuclear extracts from ATDC5 cells harboring the empty LXSN retroviral vector (lane 1), T7-TASR-1 (lane 2), or T7-TASR-2 were separated on a 12.5% SDS-polyacrylamide gel. The proteins were blotted with a mouse monoclonal anti-T7 antibody, and their positions are indicated to the right.

Fig. 2. Effects of TASR proteins on the alternative splicing of endogenous COL2 transcripts

(A). Schematic of mouse COL2 transcripts. (B) RNAs were obtained from the ATDC5 cells harboring the empty retroviral vector (top panels), T7-TASR-1 (middle panels) or T7-TASR-2 (bottom panels). RT-PCR analysis of COL2 transcripts were performed using samples from cells cultured without insulin (lanes 1–6) or with insulin (lanes 7–12). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to demonstrate that similar amounts of RNAs were present in these samples.

Fig. 3. Effects of TASR proteins on the alternative splicing of endogenous COL11A2 transcripts

(A). Schematic of mouse COL11A2 transcripts. (B) RNAs were obtained from ATDC5 cells harboring the empty retroviral vector (top panel), T7-TASR-1 (middle panel) or T7-TASR-2 (bottom panel). RT-PCR analysis of COL11A2 transcripts were performed using samples from cells cultured without insulin (lanes 1–6) or with insulin (lanes 7–12).

Fig. 4. Immunohistostaining of mouse articular cartilage with anti-TASR antibodies

Sections of normal mouse knee joint were incubated with the rabbit anti- TASR-1 (A), anti-TASR-2 (B), pre-immune IgG (C) or stained with hematoxylin–eosin (D). After several washes with PBS, the sections were incubated with 1:200 dilution of Cy3-conjugated Goat Anti-Rabbit IgG, and protein expression was examined under a fluorescence microscope. Joint surface is indicated by arrows. Bars, 20μm.