The Mechanism of Translational Repression by Iron Regulatory Protein

Gray, Nicola Kemp (1994) The Mechanism of Translational Repression by Iron Regulatory Protein. PhD thesis, University of Glasgow.

Abstract

The regulation of the intracellular iron storage protein ferritin is a cytoplasmic mechanism of gene regulation. Translation of ferritin mRNA is regulated by iron via mRNA-protein interactions between iron- responsive elements (IREs) and iron regulatory protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated region of ferritin mRNA and represses translation. The aim of this thesis was to investigate the mechanism of IRP-mediated translational control of IRE-containing mRNAs. To this end, cell-free translation systems were established in wheat germ extract and rabbit reticulocyte lysate which reconstitute IRE/IRP-mediated control. In these, cellular ferritin mRNA was specifically repressed in the presence of IRP, the IRE element was shown to be necessary and sufficient to mediate this repression, and an IRE with a single nucleotide deletion was not functional. However, the IRE element was shown not to efficiently repress translation when located in a cap-distal position. IRE/IRP-mediated regulation was determined to occur at the level of translation and not as a consequence of changes in mRNA stability. IRE/IRP-mediated regulation in vitro was found not to require a polyadenylated mRNA, suggesting that regulation in cells is not mediated by changes in poly (A) tail length. Recombinant IRP was found to be functionally equivalent to placental or reticulocyte IRP, and IRP was determined to be the only mammalian cell-specific protein required for regulation. The molecular mechanism underlying this translational repression was investigated in the cell-free systems by sucrose gradient analysis of complexes between IRE-containing mRNAs and ribosomal subunits and the IRE/IRP complex was shown to prevent the association of the 43S pre-initiation complex (including the small ribosomal subunit) with the mRNA. In addition, the formation of IRE/IRP complexes on the mRNA was found not to be sufficient in itself to block 43S access, but required the IRE to be located close to the cap structure, providing an explanation for the previously observed "position effect". Evaluation of other newly identified mRNAs containing putative IREs using these cell- free systems provided evidence that erythroid 5-aminolevulinate synthase (eALAS) and mitochondrial aconitase mRNA are also regulated by the same IRP-mediated mechanism. Furthermore, an unrelated mRNA/protein complex formed between the spliceosomal U1A protein and an indicator mRNA containing the U1A binding site, which had previously been implicated in steric repression of translation, was also shown to inhibit 43S association when located in a cap-proximal position. Based on the ability of cap-proximal U1A mRNA/U1A and IRE/IRP complexes to block the binding of the 43S complex to their respective mRNAs, it is suggested that both RNA/protein complexes inhibit translation by steric interference with the binding of the 43S pre-initiation complex. This work thus reveals that the cap-proximal region of eukaryotic mRNAs represents a sensitive target for translational repressor proteins to block the initiation of translation and raises the possibility that this may represent a more general mechanism of translational control.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Matthias Hentze
• Keywords: Genetics, Immunology
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-76337
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Dec 2019 09:15
• Last Modified: 19 Dec 2019 09:15
• URI: https://theses.gla.ac.uk/id/eprint/76337