Abstract
Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.
Highlights
The best characterized mechanisms of mRNA regulation are changes in translation where ribosome binding is the regulatory target, or mRNA stability, which is mediated by various cis-acting elements located in the coding sequences or in the untranslated region (UTR)1 of the messages [1]
We show 1) equivalent IRP1 and IRP2 binding to multiple transferrin receptor (TfR)-iron-responsive elements (IREs) RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)2 binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated)
The transferrin receptor (TfR) mRNA provides an example of an mRNA that contains both specific stability elements, the iron-responsive elements (IREs) [2,3,4,5] and AU-rich instability elements, that are common to a large family of short-lived mRNAs in
Summary
The best characterized mechanisms of mRNA regulation are changes in translation where ribosome binding is the regulatory target, or mRNA stability, which is mediated by various cis-acting elements located in the coding sequences or in the untranslated region (UTR) of the messages [1]. Volved in growth regulation and cell proliferation, such as granulocyte-macrophage colony-stimulating factor (GMC-SF) or interleukins This combination characterizes other mRNAs, such as c-myc mRNA, in this case the specific elements are localized in the coding region [1, 6, 7]. The large helix distortion created by the internal loop/bulge, unique to ferritin IRE, contributes to greater IRP2 binding relative to weak interaction of the protein with C-bulge IREs [32, 33]. Many studies of IRP interaction with different IREs, including the TfR-IREs, have used a single IRE (14,16,32– 34,36 –38) or a 3xIRE construct (IREb, IREc, and IREd) with truncated linking sequences and the less selective binding protein, IRP1 [11, 18, 39]
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