Stability of casein mRNA is ensured by structural interactions between the 3′-untranslated region and poly(A) tail via the HuR and poly(A)-binding protein complex

Abstract

The maintenance of mRNA stability has emerged as a mechanism of post-transcriptional control. We demonstrate that β-casein mRNA stability is dictated by the poly(A) tail and sequences in the 3′-UTR. An in vitro mRNA decay assay revealed that β-casein mRNA with a long poly(A) tail had higher stability than that with a short poly(A) tail. The addition of poly(A) homopolymer and 3′-UTR cRNA as competitor induced rapid degradation of β-casein mRNA. The interaction between full-length β-casein mRNA and poly(A) homopolymer was inhibited by the addition of the 3′-UTR cRNA in the lysates, which indicates that one region of the 3′-UTR associates with the poly(A) tail through an RNA–protein interaction. The putative AU-rich element (ARE) is present at nt 897–905; deletion and mutation analysis showed that the ARE site was required for maintaining the stability of the β-casein 3′-UTR. In the immunoprecipitation analysis, the poly(A)-binding protein (PABP) and the RNA-binding protein HuR were pulled down by 3′-UTR cRNA, and the absence of the ARE site reduced the binding of these proteins. These experiments further revealed that the rapid degradation of β-casein mRNA was induced by incubation with HuR- and PABP-depleted RRLs. Collectively, our results suggest that β-casein mRNA is protected from degradation by virtue of the structural interaction between the 3′-UTR and poly(A) tail via a protein complex of HuR and PABP.