S289: ppp1r1b binds iron regulatory proteins and modulates iron cellular content

Background: The IRP/IRE post-transcriptional regulatory system controls iron cellular level by modifying the expression of several proteins related to iron acquisition, mobilization and storage. This regulation is achieved by the binding of the IRP1 and IRP2 proteins to the IRE-containing UTRs in iron-deficient conditions. Previously we described the complete IRP RNP regulon system in mice (Sanchez et al 2011 Blood) identifying more than 260 mRNAs that immunoprecipitate with IRP1 and/or IRP2. Aims: Protein phosphatase 1 regulatory inhibitor subunit 1b, Ppp1r1b (encoding DARPP-32 protein) was detected as an mRNA that binds IRP1 and IRP2 in duodenum and brain. Here we focus on the characterization of the IRP/IRE regulation of the PPP1R1B gene and its role in iron metabolism. Methods: By means of EMSAs we have studied the capability of the human and mouse PPP1R1B/Ppp1r1b IRE structures to bind both IRP1 and IRP2 in vitro. Additionally, immunoblots were done to study the modulation of DARPP-32 protein by iron. Atomic absorption spectrometry was used to measure the cellular iron content in HeLa cells overexpressing the WT and phosphomutants forms of DARPP-32. Results: A 5’IRE was bioinformatically predicted in mouse Ppp1r1b mRNA. Our results indicate that the mouse Ppp1r1b 5’IRE is able to bind recombinant IRP1 in direct and competitive EMSAs in vitro. In addition, using 2 human cell lines (duodenal and neuronal) we observed iron modulation of human DARPP-32 protein in response to iron, consistently with an IRP/IRE regulation by a 5’IRE. Interestingly, overexpression of human DARPP-32 in HeLa cells caused a marked reduction in the cellular iron content, similarly as detected by Ferroportin overexpression (used as control). Using the mouse Ppp1r1b gene for overexpression in HeLa cells, a similar trend of iron depletion was found; although it did not reach statistical significance (more experiments are ongoing). We are currently investigating the molecular mechanism involved in the modulation of cellular iron content by PPP1R1B. Summary/Conclusion: The discovery of new mRNAs regulated by the IRPs will help in a better understanding of the IRP/IRE regulatory system and to unveil new functions of genes not previously linked to iron homeostasis. This study provides evidence that mouse Ppp1r1b mRNA contains a functional 5’IRE and binds IRPs in vitro. Cell culture experiments demonstrate that human DARPP-32 protein levels are regulated by iron cellular status as it would be expected for genes regulated by IRPs and we discover that DARPP-32 modulates cellular iron content. Keywords: Gene regulation, Iron, Iron metabolism