Alternative splicing of human CYR61 (CCN1) originates from intron retention and splicing pattern is altered by hypoxia

Background and aims: Alternative pre-mRNA splicing is a regulatory mechanism of gene expression to generate different protein isoforms. We investigated the Cyr61 gene expression particularly with regard to alternative pre-mRNA splicing and a potential conjunction with the strong hypoxia inducibility of this gene. Methods: Expression profiles were examined in a matched pair analysis of malignant and normal tissues samples of patients with breast or ovarian cancer (RT-PCR). Determined splice variants were cloned for sequence analysis. Hypoxia-induced alterations in alternative splicing patterns of the Cyr61 gene were analyzed in differnt human tumor cell lines. Results: The Cyr61 gene is subject to alternative splicing in cancerous and non-pathological breast and ovarian tissue. 2 different splicing variants were identified. Alternative splicing originates from intron retention of intron 3 of the human Cyr61 gene. The 131bp intron sequence includes 2 stop codons. In vitro hypoxia entailed a significant shift in alternative splicing for the benefit of the intron skipping transcript, presumably the solely mRNA variant generating a functional Cyr61 protein. Hypoxia acts as an On/Off-switch for the skipping of intron 3 that is retained under normal oxygen conditions. Conclusion: This study distinguishes the Cyr61 pre-mRNA as having 2 different alternatively spliced mRNA transcripts, whereas only the intron skipping variant seems to be capable of coding for an efficient protein. Our results suggest that oxygen deficiency presumably leads to an increased expression of functional Cyr61 protein acting as a furtherer for tumor angiogenesis and neovascularization.

Cyr61 - alternative splicing - breast cancer - hypoxia - ovarian cancer