Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3′ UTR and does not involve poly(A) tail shortening

The stability of transferrin receptor (TfR) mRNA is regulated by iron availability. When a human plasmacytoma cell line (ARH-77) is treated with an iron source (hemin), the TfR mRNA is destabilized and a shorter TfR RNA appears. A similar phenomenon is also observed in mouse fibroblasts expressing a previously characterized iron-regulated human TfR mRNA (TRS-1). In contrast, mouse cells expressing a constitutively unstable human TfR mRNA (TRS-4) display the shorter RNA irrespective of iron treatment. These shorter RNAs found in both the hemin-treated ARH-77 cells and in the mouse fibroblasts are shown to be the result of a truncation within the 3′ untranslated regions of the mRNAs. The truncated RNA is generated by an endonuclease, as most clearly evidenced by the detection of the matching 3′ endonuclease product. The cleavage site of the human TfR mRNA in the mouse fibroblasts has been mapped to single nucleotide resolution to a single-stranded region near one of the iron-responsive elements contained in the 3′ UTR. Site-directed mutagenesis demonstrates that the sequence surrounding the mapped endonuclease cleavage site is required for both iron-regulated mRNA turnover and generation of the truncated degradation intermediate. The TfR mRNA does not undergo poly(A) tail shortening prior to rapid degradation since the length of the poly (A) tail does not decrease during iron-induced destabilization. Moreover, the 3′ endonuclease cleavage product is apparently polyadenylated to the same extent as the full-length mRNA.