Investigating alternative splicing during cartilage differentation taking a transcriptomic approach

Alternative splicing (AS) of pre-mRNA transcripts is a co-transcriptional mechanism altering the protein isoforms available for translation, thus diversifying the function of the proteome of a given gene during biological processes. Involvement of AS is critical to the differentation of many human tissue types including brain and blood lines, but AS during cartilage differentiation has not yet fully been elucidated. Using transcriptomic-based bioinformatics, we aim to find out the involvement of AS in cartilage differentiation, through a combination of RNA-seq data analysis in silico paired with molecular biology and cell culture techniques. Using existing cartilage-related RNA-seq datasets, splicing analysis using command line tool SUPPA2 identified multiple splice event changes to be occurring during chondrogenesis, of which a selection were validated using mesenchymal stem cell differentiation into chondrocytes using PCR-based splice assays. Due to the observation of a known regulatory splice event occurring to splicing factor Serine And Arginine Rich Splicing Factor 3 (SRSF3) during chondrogenesis, this gene’s role was investigated using siRNA knockdown in a chondrocyte-like cell line, with its downstream target effects being confirmed by PCR splice assays. Thus far, the occurrence of AS to multiple transcription factors and splicing factors suggests its importance during chondrogenesis, and SRSF3 potentially plays an imperative role to AS regulation, with further investigation needed using SRSF3 knockdown prior to chondrogenesis, paired with functional studies to investigate the phenotypic effect on chondrogenesis.