A Conserved cis-Acting Element in the Parathyroid Hormone 3′-Untranslated Region Is Sufficient for Regulation of RNA Stability by Calcium and Phosphate

Rachel Kilav,Justin Silver,Tally Naveh-Many

doi:10.1074/jbc.m005471200

Rachel Kilav, Justin Silver + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m005471200

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Abstract

Calcium and phosphate regulate parathyroid hormone (PTH) gene expression post-transcriptionally by changes in protein-PTH mRNA 3'-untranslated region (UTR) interactions, which determine PTH mRNA stability. We have identified the protein binding sequence in the PTH mRNA 3'-UTR and determined its functionality. The protein-binding element was identified by binding, competition, and antisense oligonucleotide interference. The sequence was preserved among species suggesting its importance. To study its functionality in the context of another RNA, a 63-base pair cDNA PTH sequence was fused to the growth hormone (GH) gene. There is no parathyroid (PT) cell line and therefore an in vitro degradation assay was used to determine the stability of transcripts for PTH, GH, and a chimeric GH-PTH 63 nucleotides with PT cytosolic proteins. The full-length PTH transcript was stabilized by PT proteins from rats fed a low calcium diet and destabilized by proteins from rats fed a low phosphate diet, correlating with PTH mRNA levels in vivo. These PT proteins did not affect the native GH transcript. However, the chimeric GH transcript was stabilized by low calcium PT proteins and destabilized by low phosphate PT proteins, similar to the PTH full-length transcript. Therefore, we have identified a PTH RNA-protein binding region and shown that it is sufficient to confer responsiveness to calcium and phosphate in a reporter gene. This defined element in the PTH mRNA 3'-UTR is necessary and sufficient for the regulation of PTH mRNA stability by calcium and phosphate.

Highlights

Parathyroid hormone (PTH)1 acts to maintain serum calcium within a narrow physiological range [1]
To identify the protein binding sequence in the parathyroid hormone (PTH) mRNA 3Ј-untranslated region (UTR) we analyzed the binding of PT proteins to smaller RNA transcripts of the PTH mRNA 3Ј-UTR (Fig. 1A)
Transcripts of 30 and 26 nt formed only the smaller protein-RNA complexes with or without treatment with RNase T1 (Fig. 1B) and the larger RNA-protein complex was not formed. These results show that a transcript of 40 nt was necessary for the formation of the larger protein-RNA complex that was obtained when the full-length PTH mRNA 3Ј-UTR transcript was analyzed

Summary

EXPERIMENTAL PROCEDURES

Animals—Weanling male Sabra rats were fed a normal calcium (0.6%), normal phosphate (0.3%) diet; a low calcium (0.02%), normal phosphate (0.3%) diet; or a low phosphate (0.02%), normal calcium (0.6%) diet (Teklad, IL) for 3 weeks. RNA Electrophoretic Mobility Shift Assays (REMSA)—Labeled RNA transcripts (10,000 cpm) spanning different regions of the PTH 3Ј-UTR RNA were incubated with S100 thyroparathyroid extracts (10 ␮g), in a final volume of 20 ␮l containing 4 ␮g of tRNA, 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 5% glycerol, and 1 mM DTT (binding buffer) for 10 min at at 4 °C. UV Cross-linking Assay—UV cross-linking assays were performed as previously described [9] using 10 ␮g of S100 thyroparathyroid extracts and 32P-labeled RNA transcripts of the full-length or parts of the 3ЈUTR of the PTH cDNA in a buffer containing 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 1 mM DTT, and 5 mg/ml heparin to eliminate nonspecific binding. They were analyzed by in vitro degradation with PT proteins

RESULTS

Rat Murine Human Canine

DISCUSSION