Abstract
CD44 is a transmembrane glycoprotein involved in cell-cell and cell-matrix interactions. Several CD44 protein isoforms are generated in human through alternative splicing regulation of nine variable exons encoding for the extracellular juxta-membrane region. While the CD44 splicing variants have been described to be involved in cancer progression and development, the regulatory mechanism(s) underlying their production remain unclear. Here, we identify Tra2β and SRSF9 as proteins with opposite roles in regulating CD44 exon v10 splicing. While Tra2β promotes v10 inclusion, SRSF9 inhibits its inclusion. Mechanistically, we found that both proteins are able to target v10 exon, with GAAGAAG sequence being the binding site for Tra2β and AAGAC that for SRSF9. Collectively, our data add a novel layer of complexity to the sequential series of events involved in the regulation of CD44 splicing.
Highlights
Pre-mRNA splicing is a molecular process by which a newly made precursor messenger RNA transcript is transformed into a mature messenger RNA
Opposite to the function of Tra2β, we found that SRSF9 inhibited v10 exon inclusion significantly in both HEK293T (~30%, lane 3) and HCT116 cells (~31%, lane 6)
In both experiments, control plasmid did not affect v10 exon splicing (Figure 2A,B). These results indicate that Tra2β and SRSF9 regulate CD44 v10 exon splicing with opposite effects on v10 exon inclusion
Summary
Pre-mRNA splicing is a molecular process by which a newly made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). Variant CD44 form (CD44v) is produced by alternative splicing that comprise 10 constitutive exons and any combination of variable exons [11]. We applied a minigene-based approach to determine the opposite roles of Tra2β and SRSF9 on CD44 v10 splicing. Both proteins functionally target v10 exon, with Tra2β targeting the GAAGAAG sequence and SRSF9 targeting the AAGAC sequence located at CD44 exon v10. The ability of these RNA sequences to interact with Tra2β or SRSF9 proteins and the ability of these proteins to be the functional regulator of CD44 exon v10 splicing are well correlated.
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