Co‐transcriptional regulation during chondrogenesis

Abstract

Alternative splicing of precursor mRNA (pre-mRNA) is the major mechanism that generates diversity of protein-coding transcripts in the cell. One of the most important pre-mRNA alternative splicing events in cartilage biology involves the type II collagen gene (COL2A1). Chondroprogenitor cells synthesize exon 2-containing transcripts (type IIA and IID isoforms) while differentiated chondrocytes predominantly synthesize COL2A1 transcripts devoid of exon 2 (type IIB). We have identified another COL2A1 transcript (type IIC) generated by an alternative 5′ splice site within exon 2. In vitro studies have shown that production of the type IIC isoform is required to regulate levels of the other COL2A1 protein-coding transcripts. We are currently generating knock-in transgenic mice with specific mutations in exon 2 splice sites to directly address the role of Col2a1 isoforms in vivo during skeletogenesis. Utilizing a novel real time PCR-based method, absolute levels of the four COL2A1 isoforms in mesenchymal stem cells (MSCs) at various time-points during TGFβ3-induced differentiation was determined. Of note, the ratio of IIA/IID:IIB transcripts varied between experiments, reflecting donor-to-donor variability of MSCs. Also, when MSCs were cultured in the presence of poly(ethylene glycol) microspheres, the ratio of IIA/IID:IIB isoforms was higher compared to MSCs cultured without microspheres. This suggests that cells may respond to a biomaterial environment by synthesizing an immature collagen matrix; these findings are particularly relevant to the field of cartilage tissue engineering.