Investigating the Nuclear Roles of Iron Regulatory Protein-1A (IRP1A) in Drosophila melanogaster
Creators: Yan, Minyi
Publisher: name: University of Alberta Library
Year: 2023

<p>Iron is an indispensable micronutrient for almost all organisms. Either the lack or excess of iron can result in various human diseases. Therefore, to maintain cellular iron homeostasis, the IRP/IRE (iron regulatory protein/iron responsive element) system tightly regulates iron concentrations. Under conditions of iron depletion, IRP1 binds to the IRE located in mRNAs, thereby enhancing or inhibiting the translation of iron-related proteins. This binding leads to a net increase of iron availability within cells. When iron levels are sufficient, IRP1 incorporates an iron-sulfur cluster, referred to as holo-IRP1, which has aconitase activity and is unable to bind RNA. Considering the RNA-binding and cytosolic aconitase activity, IRP1 was believed to exclusively localize to the cytoplasm ever since its discovery ~30 years ago. However, recent studies have revealed tissue-specific nuclear localization of fly and human IRP1. In addition, further investigation via RNA-Seq analysis demonstrated that overexpression of IRP1 preferentially downregulated iron- and ecdysone-related genes in Drosophila. Given these unexpected discoveries, which challenged the traditional IRP/IRE paradigm, it is of great interest to explore the nuclear roles of IRP1A.<br>
Taking advantage of the CUT &amp; Tag methodology, I generated a chromatin-binding profile for Drosophila IRP1A, revealing distinct binding patterns in the prothoracic gland (PG) and fat body chromatin. Additionally, I found that the target genes of IRP1A in these two tissues fell into different functional categories. In the PG, IRP1A exhibited a tendency to bind to loci encoding homeotic genes (e.g., fushi tarazu, engrailed, and drumstick). In the fat body, on the other hand, IRP1A showed associations with genes related to ecdysone response and pupation, including EcR (ecdysone receptor), Eip75B (Ecdysone-induced protein 75B), broad, Fbp1 (fat body protein 1), Lsp1 (larval serum protein 1), and Blimp-1. This implies that IRP1A potentially plays a role in ecdysone signaling and the pupation process.  Furthermore, I characterized the interactomes of IRP1A and IRP1B for the PG. IRP1A appeared to interact with chromatin remodeler complexes such as Moira (MOR) and Posterior Sex Combs (Psc), suggesting its potential involvement in the maintenance of active and repressed states of homeotic genes via the Trithorax group (TrxG) and Polycomb group (PcG) proteins, respectively. Additionally, this may also imply that IRP1A indirectly influences the transcript profile of iron/ecdysone-related genes via affecting the expression of homeotic genes. Both IRP1A and IRP1B were found to bind to spliceosomal proteins Imp (IGF-II mRNA-binding protein) and CG10333, suggesting that they may affect the transcriptome via their interactions with the spliceosome machinery. To further investigate the functions of IRP1A and IRP1B, I generated CRISPR knock-in and knock-out lines, which, for example, would allow to repeat the CUT &amp; Tag analysis with endogenously controlled genes rather than transgenes. Moreover, I will compare the significance of cytosolic and nuclear of IRP1A, explore the essentiality of conformational interconversion in IRP1A, as well as identify the role of IRP1B in iron metabolism via assessing the survival rate of IRP1A/B mutants.</p>