Related Experiment Videos
Alternative RNA splicing affects function of encoded platelet-derived growth factor A chain
Abstract:
Platelet-derived growth factor (PDGF) is a basic protein of relative molecular mass 30,000 (Mr 30K) composed of two polypeptide chains, designated PDGF A and PDGF B. The B-chain is encoded by the c-sis gene, the cellular counterpart of the simian sarcoma virus transforming gene v-sis. The PDGF A-chain cDNA clones recently isolated and sequenced from a transformed human clonal glioma cell line represent at least two alternatively spliced transcript species differing by 69 base pairs at the C-terminus. Here we demonstrate that the normal human umbilical vein endothelial cell (EC) A chain precursor lacks the 15 carboxy-terminal, highly basic amino acids encoded by the larger tumour cell cDNA. Surprisingly, culture media from monkey kidney cells (COS) transfected with the endothelial cDNA clone contained much less mitogenic activity than media from cells transfected with the longer tumour cell-derived A-chain cDNA. This functional difference appeared to be due to inefficient assembly or secretion of the recombinant endothelial-type growth factor. This suggests that some transformed cells may use alternative RNA splicing to modify normal growth factors and by so doing increase the efficiency of mitogen assembly or secretion.
Insights
Platelet-derived growth factor (PDGF) A-chain variants show differential mitogenic activity. Alternative RNA splicing in tumor cells may enhance growth factor secretion efficiency.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Platelet-derived growth factor (PDGF) is a mitogenic protein crucial for cell growth.
- PDGF consists of A and B polypeptide chains, with the B-chain encoded by the c-sis gene.
- Alternative splicing of PDGF A-chain transcripts occurs in transformed cells.
Purpose of the Study:
- To investigate the functional differences between normal and tumor-derived PDGF A-chain variants.
- To determine the impact of alternative RNA splicing on PDGF secretion and mitogenic activity.
Main Methods:
- Isolation and sequencing of PDGF A-chain cDNA from endothelial cells and glioma cells.
- Transfection of monkey kidney cells (COS) with different PDGF A-chain cDNA constructs.
- Measurement of mitogenic activity in cell culture media.
Main Results:
- Normal human umbilical vein endothelial cell (EC) A-chain precursor lacks specific C-terminal amino acids found in tumor cell cDNA.
- Culture media from COS cells transfected with endothelial cDNA showed significantly lower mitogenic activity compared to those transfected with tumor cell cDNA.
- Inefficient assembly or secretion of the endothelial-type growth factor likely accounts for the reduced activity.
Conclusions:
- Alternative RNA splicing of PDGF A-chain in transformed cells can alter growth factor properties.
- Tumor cells may utilize alternative splicing to enhance the efficiency of growth factor assembly and secretion.
- This mechanism could contribute to uncontrolled cell proliferation in cancer.