KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and stability in transfected cells.

BACKGROUND: Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally through the binding of the trans-acting proteins AU rich binding factor 1 (AUF1), Upstream of N-ras (Unr) and KH-type splicing regulatory protein (KSRP) to an AU rich element (ARE) in PTH mRNA 3'-UTR. AUF1 and Unr stabilize PTH mRNA while KSRP, recruiting the exoribonucleolytic complex exosome, promotes PTH mRNA decay. RESULTS: PTH mRNA is cleaved by the endoribonuclease polysomal ribonuclease 1 (PMR1) in an ARE-dependent manner. Moreover, PMR1 co-immunoprecipitates with PTH mRNA, the exosome and KSRP. Knock-down of either exosome components or KSRP by siRNAs prevents PMR1-mediated cleavage of PTH mRNA. CONCLUSION: PTH mRNA is a target for the endonuclease PMR1. The PMR1 mediated decrease in PTH mRNA levels involves the PTH mRNA 3'-UTR ARE, KSRP and the exosome. This represents an unanticipated mechanism by which the decay of an ARE-containing mRNA is facilitated by KSRP and is dependent on both the exosome and an endoribonuclease.

Identification and characterization of cis-acting elements in the human and bovine PTH mRNA 3'-untranslated region.

UNLABELLED: 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, Ca(2+) 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.

The protein phosphatase calcineurin determines basal parathyroid hormone gene expression.

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.

The parathyroid hormone mRNA 3'-untranslated region AU-rich element is an unstructured functional element.

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.

Cis and trans acting factors in the regulation of parathyroid hormone (PTH) mRNA stability by calcium and phosphate.

Calcium and phosphate regulate parathyroid hormone (PTH) mRNA stability through differences in binding of parathyroid proteins to an element in its 3'-untranslated region. One of the proteins is AUF1 (A+U-rich element binding factor 1). An in vitro degradation assay showed that transcripts for PTH and chimeric growth hormone (GH)-PTH 63 nt, but not for native GH, were stabilized by PT proteins from rats on low calcium diets and destabilized by proteins from rats on low phosphate diets, correlating with PTH mRNA levels in vivo. In transfection experiments the 63 nt binding element destabilized mRNAs of reporter genes and this was prevented by over-expression of AUF1. Our results identified a functional cis element in PTH mRNA. Differences in protein binding to this element determine PTH mRNA stability and its regulation by calcium and phosphate.