Interaction between 3′ untranslated region of calcitonin receptor messenger ribonucleic acid (RNA) and adenylate/uridylate (AU)-rich element binding proteins (AU-rich RNA-binding factor 1 and Hu antigen R)

Abstract

Our previous results in mouse osteoclasts suggested that calcitonin (CT) alters CT receptor (CTR) mRNA stability. The CTR mRNA transcript contains several adenylate/uridylate (AU)-rich destabilizing elements in the 3′ untranslated region (3′UTR). When the 3′UTR of mouse CTR mRNA was labeled by [α-32P]-uridine 5-triphosphate, interactions were observed between the transcript and several cell extracts, including those from the osteoclast progenitor monocyte/macrophage cell line, RAW 264.7. The molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to AU-rich RNA-bindiug factor 1 (AUF1) and Hu antigen R (HuR). Radiolabeled 3′UTR transcripts bound with a 40-kDa protein, which could be extracted from cells transfected with AUF1 p40. To confirm the binding specificity, a pSG5 vector construct, containing the AUF1 p40 with an hemagglutinin tag, was transiently transfected into NIH3T3 cells. The extracts were incubated with poly(A)-added CTR3′UTR, The reaction mixture was immunoprecipitated using an antihemagglutinin antibody and precipitated mRNA species were extracted and reverse transcribed using oligo-dT primers. It was found that PCR primers specific for the 3′UTR of CTR mRNA sequence generated a PCR signal. No signal was observed when mutated AUF1 p40 was transfected. In a manner similar to the AUF1 binding, HuR was also found to bind to the 3′UTR. Specific binding of AUF1 p40 and HuR was also found with RNA extracted from mouse osteoclasts. Treatment of osteoclasts with CT did not significantly affect the expression of AUF1 but decreased the levels of HuR and its mRNA. The role of CTR3′UTR in mRNA stability was further tested by expressing luciferase reporter constructs that did, or did not, contain the CTR3′UTR, under the control of the tetracycline-regulatory system. The results showed that the addition of 3′UTR considerably shortened the mRNA half-life of the luciferase reporter gene. These results suggest that AUF1 p40, HuR, and the 3′UTR of the CTR mRNA transcript could be involved in posttranscriptional regulation of CTR mRNA expression.