Abstract
The long and GC-rich 5'-untranslated region (5'-UTR) of the known 3.8-kb platelet-derived growth factor B (PDGF-B)/c-sis mRNA is highly conserved and inhibits its own translation. It has been thought that this 5'-UTR functions by regulating translation possibly using an internal ribosome entry site (IRES)-mediated mechanism. However, in the present study we found no evidence that the 5'-UTR sequence of PDGF-B mRNA contains any IRES activity. Instead, we found that the 5'-UTR sequence of PDGF-B functions as a promoter both constitutively and upon induction in a variety of cell lines. The 5'-UTR sequence contains two promoters (termed P1 and P2) when only the 5'-UTR sequence is analyzed. In the presence of the upstream TATA-box-containing promoter (P0), P1 and P0 promoters are integrated into one promoter, whereas the P2 promoter still functions. The full promoter with combined P0, P1, and P2 produced two transcripts, with the major one having the full-length 5'-UTR and the minor one the short 5'-UTR. The integrated P0/P1 promoter and P2 promoter are likely responsible for producing the endogenous 3.8- and 2.8-kb PDGF-B mRNAs that are detected in cultured human renal microvascular endothelial cells, a few tumor cells, and rat brain tissues. Furthermore, we detected the 2.8-kb PDGF-B mRNA in erythroleukemia K562 cells upon 12-O-tetradecanoylphorbol-13-acetate-induced differentiation. Considering that the 5'-UTR in the 3.8-kb mRNA contains no IRES activity and inhibits cap-dependent translation, we believe that the endogenous 2.8-kb mRNA produced from the 5'-UTR promoter is likely the major template responsible for protein production both constitutively and upon induction. We also found that the transcription from the 5'-UTR P2 promoter might be coordinated by the major upstream P0 promoter upon stimulation. Based on these observations, we propose that the TATA-containing P0 promoter and the 5'-UTR promoter work together to tightly control the expression of PDGF-B.
Highlights
Platelet-derived growth factor B (PDGF-B)1/c-sis belongs to a family of proteins consisting of four gene products, PDGF-A, platelet-derived growth factor B (PDGF-B), PDGF-C, and PDGF-D
The 5Ј-truncated PDGF-B mRNA was observed in a few tumor cell lines and increased in rat brain at a certain stage of brain development, and the level of the 5Ј-truncated mRNA was shown to be associated with PDGF-B protein level [12]
In this study, using firefly luciferase as a heterologous reporter gene, we found that the full-length 5Ј-untranslated region (5Ј-UTR) of PDGF-B inhibits translation of the luciferase gene in a rabbit reticulocyte lysate cell-free system (Fig. 2)
Summary
Cause human erythroleukemia K562 cells are differentiated into megakaryocytes upon TPA stimulation, regulation of PDGF-B expression in K562 cells has been extensively studied. We report our surprising finding that the 2.8-kb human PDGF-B mRNA, previously detected in HRMECs [13], is produced in K562 cells upon TPA-induced differentiation This transcript represents a minor species in Northern blot analysis. Using luciferase reporter promoter assay and Northern blot analysis, we demonstrated that the DNA sequence encoding the 5Ј-UTR of the long PDGF-B mRNA contains promoters that function in various cell lines and produces two mRNA species with a medium and a short 5Ј-UTR, respectively. Based on the above observations, we conclude that the major TATA-containing promoter and the promoters in the 5Ј-UTR work together to control the expression of the 2.8-kb PDGF-B transcript in a variety of cells as an effective source of mRNA for protein production. Tight control of the production of the 2.8-kb mRNA and its stability may be used widely to control PDGF-B expression, both constitutively and upon stimulation
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