Identification and Characterization of cis-Acting Elements in the Human and Bovine PTH mRNA 3′-Untranslated Region

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Abstract
The human PTH mRNA 3′‐UTR has a cis element homologous to the rat cis‐acting instability element and a more proximal element identical to the single binding element identified in bovine PTH mRNA 3′‐UTR. The function of the elements was shown in vitro. Introduction: In the rat, Ca2+ and phosphate regulate PTH mRNA stability by the interaction of trans‐acting proteins with a defined cis‐acting instability element in the distal region of the PTH mRNA 3′‐untranslated region (UTR). This element has been characterized in the rat and is conserved in human, canine, feline, and murine 3′‐UTRs but not in bovine and porcine 3′‐UTRs. Materials and Methods: Parathyroid protein‐binding assays to the PTH mRNA transcripts were performed. Functionality was studied in reporter genes that were transiently transfected into HEK293 cells. Results: Protein‐RNA binding experiments identified an element in bovine PTH mRNA at the proximal end of the 3′‐UTR that is different from the rat protein‐binding element. The human 3′‐UTR contains both elements, but only the distal element binds proteins. Functional studies with HEK293 cells transiently transfected with reporter genes containing the different elements and flanking nucleotides (nt) showed that the human distal element destabilized a reporter mRNA similar to the effect of this element in the rat. A reporter mRNA containing the single bovine PTH mRNA protein‐binding element was also destabilized, and this was prevented by coexpression of AU‐rich element binding factor 1 (AUF1). Conclusion: Our results identify a new protein‐binding element in the PTH mRNA 3′‐UTR. In bovine PTH mRNA, it is the only element, and it is functional in destabilizing a reporter gene. It is also present in other species, including human PTH mRNA, where it is not functional, possibly because of differences in flanking sequences. The human PTH mRNA 3′‐UTR distal element is highly homologous to the rat cis‐acting instability element and destabilized a reporter gene, indicating its functionality. Therefore, different species have alternative cis‐acting protein‐binding elements that may determine the regulation of PTH mRNA stability in response to changes in serum calcium and phosphate.