Abstract
We have previously demonstrated that phorbol myristate acetate (PMA) up-regulates H-ferritin gene expression in myeloid cells by stabilization of its message. In the present report, we showed that insertion of the 3'-untranslated region (3'-UTR) of H-ferritin mRNA at the 3'-end of luciferase coding sequence significantly reduced the stability of luciferase mRNA in human monocytic THP-1 cells. However, the half-life of the chimeric transcript was markedly prolonged after PMA treatment. A cytosolic protein factor from THP-1 cells was found to specifically bind to H-ferritin 3'-UTR. PMA treatment of THP-1 cells resulted in the reduction of the RNA binding activity in a time-dependent manner. Deletion analysis and RNase T1 mapping revealed a pyrimidine-rich sequence within the 3'-UTR which interacts with the protein factor. Competition experiments with homoribopolymers further demonstrated the importance of uridines for the binding activity. Point mutations in uridines of the pyrimidine-rich sequence reduced the protein binding to 3'-UTR, while increasing the stability of the chimeric luciferase transcript. Together, these results demonstrate that the pyrimidine-rich sequence in the 3'-UTR is involved in post-transcriptional regulation of H-ferritin gene expression in myeloid cells.
Highlights
Ferritin is a multimeric protein with a function in controlling the iron homeostasis in cells [1, 2]
Using the human monocytic THP-1 cell line as a model system, we have demonstrated that phorbol myristate acetate (PMA)-induced differentiation of THP-1 cells toward macrophages markedly up-regulates the expression of H-ferritin mRNA, but not L-ferritin mRNA, in a cell-type specific manner [16]
Since the stability of most mRNAs has been shown to be regulated by sequences in their 3Ј-UTRs [17], we hypothesized that an unique cis-regulatory element within the 3Јuntranslated region (3Ј-UTR) of H-ferritin mRNA is likely involved in regulating the stability of the H-ferritin message in PMA-treated THP-1 cells
Summary
H, heavy; L, light; PMA, phorbol 12myristate 13-acetate; 3Ј-UTR, 3Ј-untranslated region; RT-PCR, reverse transcriptase-polymerase chain reaction. Using the human monocytic THP-1 cell line as a model system, we have demonstrated that phorbol myristate acetate (PMA)-induced differentiation of THP-1 cells toward macrophages markedly up-regulates the expression of H-ferritin mRNA, but not L-ferritin mRNA, in a cell-type specific manner [16]. Since the stability of most mRNAs has been shown to be regulated by sequences in their 3Ј-UTRs [17], we hypothesized that an unique cis-regulatory element within the 3Јuntranslated region (3Ј-UTR) of H-ferritin mRNA is likely involved in regulating the stability of the H-ferritin message in PMA-treated THP-1 cells. PMA treatment down-regulated the binding of the protein factor to H-ferritin 3Ј-UTR, suggesting that the interaction between the cytosolic protein and the cis element serves as a destabilizing signal to facilitate the degradation of Hferritin mRNA in THP-1 cells
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