Genetic control of metabolism: the regulome of iron regulatory proteins
Final Report Abstract
Metabolic remodeling is crucial for cellular functions. Genetic control of metabolism is best understood at the level of transcription, yet the role of posttranscriptional processes in metabolic regulations is ill understood. Iron metabolism is ideally suited to study the posttranscriptional regulation of metabolism. Iron metabolism is regulated by two RNA binding factors named iron regulatory proteins (IRP)-1 and -2, which interact with cis-regulatory stem-loop structures called iron responsive elements (IRE) and thereby modulate the translation or stability of target mRNAs. Our current knowledge of the IRP regulome is limited to 8 IRE-containing genes encoding iron metabolism molecules. However, multiple evidence suggests that the regulatory scope of the IRP/IRE system is wider, and that yet unknown IRP targets are to be discovered. In this work, we integrated several omics technologies to address 3 main questions: I) how many and which mRNAs bear IRP-binding sites (IRP RNA interactome), II) what are the translational targets of the IRPs (IRP translatome), III) which mRNAs are regulated by the IRPs at the level of their turnover (IRP RNA stabilome). Using eCLIP (enhanced UV cross-linking and immuno-precipitation) technology, we were able to map 1371 high-confidence IRP1-binding regions in the transcriptome of hepatic cells, including all known IRE-containing transcripts. Most IRP1 binding sites occur as single entities and are principally present in protein-coding transcripts, with 1109 IRP1-RNA interactions detected in 989 mRNAs. IRP1-mRNA contacts are mainly located in coding regions (CDS) and in 3’ untranslated regions (UTR), with less than 10% of IRP1 binding sites being present in 5’UTRs. A computational analysis revealed stem-loop structures resembling the „canonical“ IRE in 36 RNA sequences bound by IRP1. This suggests that the known IRP-IRE interaction is rather an exception, and that the majority of IRP-RNA contacts involves distinct modes on interaction. A preliminary search for RNA interaction motifs across all IRP1- binding sites started to uncover novel RNA structures possibly mediating the interaction with IRP1. We used ribosome profiling to characterize the fraction of the transcriptome that is controlled by the IRPs at the translational level. We sequenced ribosome footprints from normal cells versus cells ablated for both IRP1 and IRP2. IRP deficiency alters the translation of more than 500 genes in cultured cells. In vivo, IRP ablation in hepatocytes affects the translation of a smaller portion of the liver transcriptome (about 70 mRNAs). A search for gene ontology term enrichment revealed that the IRP translatome is mostly connected to pathways involved in developmental processes or in defense mechanisms, respectively. Of note, 32 genes of the IRP translatome also bear IRP1 CLIP tags, potentially representing bona fide IRP targets controlled via direct IRP-RNA contacts. Out of 989 genes bearing IRP1-binding sites, only 32 are regulated at the translational level. This implies that IRP-RNA contacts mostly affect other aspects of RNA metabolism, such as RNA decay. To identify the mRNAs whose turnover is regulated by the IRPs, we co-ablated IRP1 and IRP2 ex vivo in cultured cells or in vivo in mouse liver, respectively, and analyzed the ensuing changes in RNA halflives using SLAM-Seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA). This recent technique is based on metabolic labeling of nascent RNA with 4-thio-uridine (4sU), a nucleoside analogue that can be chemically modified to generate thymine to cytosine conversions (T>C) during RNA sequencing. Data computing is still ongoing, but started to reveal interesting feaures, with for example a positive effect of the IRPs on the stability of an mRNA bearing a novel IRE motif and encoding a protein involved in vesicle-mediated transport. Our multi-omics approach enables us to explore the regulatory scope of the IRP network in unprecedented depth. Although not yet complete, our work already uncovered many potential new targets, identified within the natural cellular environment. It also suggests the existence of novel modes of interaction between the IRPs and their mRNA targets. I believe our study could serve as benchmark for similar studies aimning at deciphering the regulatory lanscape of protein which, alike the IRPs, bind RNA in an unconventional manner. Our work also paves the way towards novel discoveries, from the structural determinants of the newly identified IRP-RNA interactions to their role in physiology and disease.
Publications
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(2016) Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure. Eur Heart J., 38:362-72
Haddad S, Wang Y, Galy B, Korf-Klingebiel M, Hirsch V, Baru AM, Rostami F, Heineke J, Flögel U, Groos S, Renner A, Toischer K, Zimmermann F, Engeli S, Jordan J, Bauersachs J, Hentze MW, Wollert KC, Kempf T
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(2016) Mice with hepcidin-resistant ferroportin accumulate iron in the retina. FASEB J., 30:813-23
Theurl M, Song D, Clark E, Sterling J, Grieco S, Altamura S, Galy B, Hentze MW, Muckenthaler MU, Dunaief, JL
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(2017) A red carpet for iron metabolism. Cell, 168:344-61
Muckenthaler M, Hentze MW, Rivella S, Galy B
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(2017) The actin binding protein profilin 2 is a novel regulator of iron homeostasis. Blood, 130:1934-45
Luscieti S, Galy B, Gutierrez L, Reinke M, Couso J, Shvartsman M, DI Pascale A, Witke W, Hentwe MW, Pilo Boyl P, Sanchez M
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(2019) Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation. J. Lipid Res.
Von Gerichten J, Lamprecht D, Opálka L, Soulard D, Marsching C, Pilz R, Herzer S, Galy B, Nordström V, Hopf C, Trottein F, Sandhoff R
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(2019) Effects of the Antifungal Agent Ciclopirox in HPV-Positive Cancer Cells: Repression of Viral E6/E7 Oncogene Expression and Induction of Senescence and Apoptosis. Int. J. Cancer
Braun J, Hermann AL, Blase JI, Frensemeier K, Bulkescher J, Scheffner M, Galy B, Hoppe-Seyler K, Hoppe- Seyler F
