Related Experiment Videos
Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3'
R Binder1, J A Horowitz, J P Basilion
1Cell Biology and Metabolism Branch, NICHD, NIH, Bethesda, MD 20892.
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.
Insights
Iron regulates transferrin receptor (TfR) mRNA stability. An endonuclease cleaves TfR mRNA in its 3' untranslated region, generating a shorter, unstable RNA fragment in response to iron levels.
Area of Science:
- Molecular Biology
- Gene Regulation
- RNA Metabolism
Background:
- Transferrin receptor (TfR) mRNA stability is a key regulator of cellular iron uptake.
- Iron availability influences TfR mRNA levels, but the precise mechanism of destabilization is not fully understood.
Purpose of the Study:
- To investigate the mechanism of iron-regulated TfR mRNA destabilization.
- To identify the specific site and process of TfR mRNA cleavage.
Main Methods:
- Cell culture (ARH-77, mouse fibroblasts) with iron treatment (hemin).
- Analysis of TfR mRNA and RNA fragments using molecular biology techniques.
- Mapping of mRNA cleavage sites and site-directed mutagenesis.
Main Results:
- Iron treatment of ARH-77 cells and mouse fibroblasts expressing TfR mRNA leads to a shorter TfR RNA.
- This shorter RNA results from endonuclease cleavage within the 3' untranslated region (UTR).
- The cleavage site is mapped to a single nucleotide within a single-stranded region near an iron-responsive element in the 3' UTR, and this sequence is crucial for regulation.
Conclusions:
- Iron-regulated TfR mRNA decay is mediated by an endonuclease that cleaves the mRNA within the 3' UTR.
- The specific sequence surrounding the cleavage site is essential for iron-dependent mRNA turnover.
- Poly(A) tail shortening is not the primary mechanism for TfR mRNA degradation in response to iron.