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.

Abstract

The stability of transferrin receptor (TfR) mRNA is regulated by iron availability. When a human plasma‐cytoma 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.

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat

“…When IRPs bind to TfR mRNA, they retard its degradation, and mRNA has a half-life of 6 h. When IRPs are not bound to the mRNA, it has a much shorter half-life (o1 h). 66 However, TfR2 is encoded by an mRNA that lacks IREs, which indicates that in some circumstances regulation of the number of Tf-binding sites in the cell would not be controlled by IRP. 67 In contrast, expression of TfR2 appears to be refractory to alterations in iron status, at least in liver.…”

Renal Iron Metabolism

“…When IRP1 is in the cytosolic aconitase form, it does not bind to IRE of the target transcripts, and the translation of H-and L-ferritin proceeds freely, creating a 24-subunit spherical protein that can oxidize and sequester several thousand Fe 3ϩ atoms within its core (Figure 4). TfR1 contains five IRE in its 3Ј UTR, and when IRP are not bound to the 3Ј UTR, TfR mRNA undergoes iron-dependent degradation and TfR levels decrease accordingly (32). IRP2 also binds to IRE in iron-depleted cells.…”

“…TfR changes may be difficult to assess in kidney lysates because different cell types are mixed together in lysates. TfR mRNA degradation also requires a specific but uncharacterized endonuclease that may be nonabundant in kidney (32) (Figures 1 and 5). …”

Functional Characterization of the Interaction between Human La and Hepatitis B Virus RNA

“…The requirement of several trans-acting factors was also described for interleukin-2 mRNA stability (44). The transferrin receptor mRNA is stabilized by the iron-regulatory protein, which interacts with the 3Ј-end of the RNA if cellular iron levels are low, thereby protecting the RNA against endoribonucleolytic cleavage (45). An increase in cellular iron concentration leads to reduced affinity of the iron-regulatory protein to the transferrin receptor mRNA and subsequently to the degradation of the RNA (reviewed in Ref.…”