Abstract
Cap proximity is a requirement to enable secondary structures and RNA-binding proteins to repress translational initiation via the 5'-untranslated region (5'-UTR) of mammalian mRNAs. We show that in Saccharomyces cerevisiae, unlike mammalian cells, the in vitro translational repressive effect of the mammalian iron regulatory protein 1 (IRP1) is independent of the site of its target in the 5'-UTR, the iron-responsive element (IRE). In vitro studies demonstrate that the binding affinity of IRP1 is also unaffected by the position of the IRE. Using IRE loop mutants, we observe an almost complete loss of IRP1-dependent repression in yeast concomitant with a 150-fold reduction in binding affinity for the IRE target. This mirrors the natural quantitative range of iron-induced adjustment of IRE/IRP1 affinity in mammalian cells. By enhancing the stability of the IRE stem-loop, we also show that its intrinsic folding energy acts together with the binding energy of IRP1 to give an additive capacity to restrict translational initiation. An IRE.IRP1 complex in a cap-distal position in yeast blocks scanning 40 S ribosomes on the 5'-UTR. It follows that the position effect of mammalian site-specific translational repression is dictated by the competence of the mammalian preinitiation complex to destabilize inhibitory structures at different steps of the initiation process.
Highlights
RNA-RNA and RNA-protein interactions are dominant features of posttranscriptional gene expression
By enhancing the stability of the iron-responsive element (IRE) stem-loop, we show that its intrinsic folding energy acts together with the binding energy of iron regulatory protein 1 (IRP1) to give an additive capacity to restrict translational initiation
The primary pathway of eukaryotic translational initiation for the majority of cellular mRNAs is currently described by a working model which envisages that 40 S ribosomal subunits progressively scan the 59-UTR1 from the 59 end in search of start codons [4]
Summary
RNA-RNA and RNA-protein interactions are dominant features of posttranscriptional gene expression. This implies that either the free energy changes of the early ribosome-mRNA interactions are smaller than the thermodynamic driving force intrinsic to the scanning process, or ribosomes can “skip” structural barriers more readily in a cap-distal position Neither of these principles seems to apply to S. cerevisiae, in which the position of a stem-loop structure within the 59-UTR is of little significance in terms of the degree of translational inhibition observed [10, 11]. The best characterized eukaryotic example of this type of regulation is based on the binding of the iron regulatory proteins (IRP1 and IRP2) to the iron-responsive element (IRE) in the 59-UTRs of the mRNAs encoding ferritin and erythroid 5-aminolevulinic acid synthase in vertebrate cells [17]. The expression of the IRP1 gene was found to be sufficient for strong translational repression of an mRNA bearing an IRE-containing 59-UTR in S. cerevisiae demonstrating that regulation requires no mammalian components other than IRP1 and IRE to function [20]
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