Abstract
Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3'-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate that the PTH mRNA 3'-UTR and in particular the region of the cis-element are dominated by significant open regions with little folded base pairing. Mutation analysis of the 26-nt core element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3'-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors.
Highlights
Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia
There was a marked decrease in growth hormone (GH) and green fluorescent protein (GFP) mRNA levels of the chimeric constructs compared with the WT constructs or chimeric constructs that contained a truncated sequence of the PTH mRNA element, and this effect was post-transcriptional
We have developed a reporter assay for the function of the PTH mRNA 3Ј-untranslated region (UTR) defined element
Summary
Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. The native GH transcript was more stable than PTH and the chimeric RNAs and was not affected by PT proteins from the different diets These results demonstrate that the protein binding region of the PTH mRNA 3Ј-UTR is both necessary and sufficient to confer responsiveness to calcium and phosphate and determines PTH mRNA stability and levels [5]. There was a marked decrease in GH and GFP mRNA levels of the chimeric constructs compared with the WT constructs or chimeric constructs that contained a truncated sequence of the PTH mRNA element, and this effect was post-transcriptional These results are in agreement with the decreased stability of the chimeric transcript in the in vitro degradation assay with PT proteins, and they demonstrate the functional importance of the RNA protein binding region in the PTH 3Ј-UTR [5]. Mutation analysis confirmed the importance of specific sequence patterns for protein binding and for the destabilizing effect of the cis-acting element
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