Position is the critical determinant for function of iron-responsive elements as translational regulators.

Abstract

At least two groups of eukaryotic mRNAs (ferritin and erythroid 5-aminolevulinate synthase) are translationally regulated via iron-responsive elements (IREs) located in a conserved position within the 5' untranslated regions of their mRNAs. We establish that the spacing between the 5' terminus of an mRNA and the IRE determines the potential of the IRE to mediate iron-dependent translational repression. The length of the RNA spacer rather than its nucleotide sequence or predicted secondary structure is shown to be the primary determinant of IRE function. When the position of the IRE is preserved, sequences flanking the IRE in natural ferritin mRNA can be replaced by altered flanking sequences without affecting the regulatory function of the IRE in vivo. These results define position as a critical cis requirement for IRE function in vivo and imply the potential to utilize transcription start site selection to modulate the function of this translational regulator.The biosynthesis of the iron storage protein ferritin and of erythroid 5-aminolevulinic acid synthase (eALAS), an enzyme involved in the major iron utilization pathway of the human body, is translationally determined by cellular iron status (1, 6, 7, 8a, 15, 17, 28, 33, 34, 40; unpublished observations). Previous work established that the interaction between an iron-responsive element (IRE) contained in the 5' untranslated region (UTR) of an mRNA and a specific cytoplasmic IRE-binding protein, IRE-BP, results in translational repression of the mRNA in vivo and in vitro (2, 4, 9, 36). Iron regulation of mRNA translation results from irondependent control of the binding activity of 18,24).The presence of an IRE in the 5' UTR of ferritin mRNA has been established as a necessary cis requirement for translational control of ferritin expression (4, 15). However, the conditions under which the presence of an IRE in the 5' UTR of an mRNA suffices for iron control are less clearly defined. When the complete 5' UTR of a ferritin cDNA was fused to human growth hormone (hGH) or chloramphenicol acetyltransferase protein-coding regions or when a synthetic oligodeoxyribonucleotide encoding a ferritin IRE was cloned into the 5' UTR of the hGH or chloramphenicol acetyltransferase gene, expression of the indicator proteins was rendered iron responsive, suggesting that the presence of an IRE in the 5' UTR was sufficient for regulation (1,9,15,17). However, introduction of one or more nonfunctional IRE mutAhts between the 5' end and the intact IRE of an hGH indicator construct renders hGH expression unresponsive to iron (9). On the basis of this finding, the posltion of the IRE was implicated as a possible critical deterhinant of IRE function. Apart from positional requirements, the regions flanking the IRE in ferritin mRNA also seemed to be functionally important (13,38). Since this suggestion was derived from in vitro studies of IRE-BP binding to ferritin mRNA, we evaluated the contribution of the flanking regions to IRE function as a translational regulator in vivo.In thi...

Ribosomal Pausing and Scanning Arrest as Mechanisms of Translational Regulation from Cap-Distal Iron-Responsive Elements

“…The possibility that the observed IRE position effect could result from different affinities of the respective IREs for IRP-1 was excluded by competition gel retardation assays (data not shown). This is consistent with previous results (10).…”

“…This study addresses a long-standing question that was raised when the positions of IREs in various mRNAs and from different species were found to be phylogenetically conserved and functionally important (9,10). Subsequent studies added further examples of cellular mRNAs that appear to obey the position effect (15,32) and also uncovered possible exceptions (15,23 [see below]).…”

“…IRE.34 mRNA in this study and F44 mRNA in the transfection experiments (10) served as positive controls and points of reference for the profound repressive effect of IRP binding to a cap-proximal IRE. IRE.66 mRNA (this study) and F64 mRNA (in the transfection experiments [10]) allowed a comparative assessment of the role of IRE-IRP complexes formed more than 60 nucleotides downstream from the cap structure. Our results show that the RRL system reflects the roughly twofold (50%) difference between F44 and F64 mRNAs in vivo (10) and IRE.34 and IRE.66 mRNAs in vitro (Fig.…”

“…An IRE placed Ͼ60 nucleotides downstream from the cap structure mediates only partial translational inhibition by IRP binding. In keeping with this position effect, the cap-proximal location of mammalian IREs is phylogenetically conserved (10). The manifestation of the position effect displays cell-type-dependent quantitative differences (10), suggesting that the ability of the translation apparatus to overcome cap-distal IRE-IRP complexes can be affected by cellular determinants.…”

“…IRP binding to the IRE of ferritin mRNAs affects an early step of translation initiation: it prevents the recruitment of the 43S translation preinitiation complex (which includes the small ribosomal subunit) (13,33). Transfection studies using mammalian tissue culture cells revealed a characteristic feature of this IRE-IRP regulatory mechanism: for IRP binding to efficiently block translation, the IRE must be located within Ͻ60 nucleotides from the m 7 GpppN-cap structure of the mRNA (9,10). An IRE placed Ͼ60 nucleotides downstream from the cap structure mediates only partial translational inhibition by IRP binding.…”