Translational repression and novel functions of cth2 in the saccharomyces cerevisiae response to iron deficiency

• Autores: Mª Lourdes Ramos Alonso
• Directores de la Tesis: María Teresa Martínez Pastor (dir. tes.), Sergi Puig Todolí (codir. tes.)
• Lectura: En la Universitat de València ( España ) en 2020
• Idioma: español
• Tribunal Calificador de la Tesis: Enrique Herrero Perpiñan (presid.), Paula Alepuz Martínez (secret.), Susana Rodríguez Navarro (voc.)
• Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
• Materias:
  • Química
    • Bioquímica
• Enlaces
  • Tesis en acceso abierto en: TESEO
• Resumen

  • Iron is an essential micronutrient for all eukaryotes because of its redox properties. It participates as a cofactor in a wide range of biological processes, including protein translation, mitochondrial respiration (Krebs cycle and electron transport chain (ETC)) and DNA replication. The model organism Saccharomyces cerevisiae responds to iron deficiency by activating the iron acquisition and recycling systems, and by remodeling cellular metabolism to promote iron utilization in specific processes at the expense of others. The tandem zinc finger (TZF)-containing protein Cth2 plays an important role in prioritizing iron by promoting the degradation of multiple mRNAs containing A/U-rich elements (AREs), including the CTH2 mRNA itself that is autoregulated. In this thesis, we identified and characterized new mechanisms involved in the global translational repression and novel functions of Cth2 in response to iron starvation. Our results with polysome fractionation experiments demonstrate that during the progress of iron deficiency the eIF2α/Gcn2 pathway is involved in the general repression of translational initiation. The Gcn2 kinase specifically phosphorylates serine 51 of eIF2α in a Gcn1-dependent manner, causing a slight induction of GCN4 translation when iron is scarce. The Gcn2 activation by uncharged tRNAs and TORC1 inactivation during the iron deficiency is discussed. Besides, we show a role played by Cth2 in translational inhibition of several ARE-containing mRNAs during iron deficiency. Both the Cth2 TZF-domain as well as the AREs within SDH4 (subunit of succinate dehydrogenase) and CTH2 mRNAs are essential for translational repression. Besides, other mRNAs are translationally repressed by Cth2, suggesting a Cth2 general role on inhibition of translation of ARE-containing mRNAs. Our results also demonstrate that while the amino-terminal domain (NTD) of Cth2 is important for both mRNA turnover and translational inhibition functions, its carboxy-terminal domain (CTD) is only involved in translational repression. Importantly, Cth2 CTD is physiologically relevant during iron-deficient conditions. Two novel Cth2 functions under iron deficiency include the regulation of mitochondrial respiration and the RNR3 catalytic subunit of the iron-dependent ribonucleotide reductase (RNR) enzyme responsible of dNTP synthesis. The overexpression of CTH2 under iron sufficiency decreases respiration (measured by the oxygen consumption) as well as several iron-dependent enzymatic activities, including Leu1, aconitase (from Krebs cycle) and complex II and III of the ETC, while the complex IV activity is unaffected. Under iron deficiency, oxygen consumption decreases regardless Cth2, despite the decrease in complex II and III activities of the ETC. Interestingly, under iron starvation Cth2 contributes to a better complex IV activity, probably by increasing Cox1 protein levels. Finally, RNR3 has been described to be highly expressed under genotoxic or replication stress conditions by the Mec1–Rad53–Dun1 checkpoint kinase pathway. However, RNR3 function is not clear as its paralog RNR1 was described to be the major isoform of the catalytic subunit of RNR. Our results suggest a higher RNR3 expression under long-term iron deficiency comparable to that observed under those stresses. Besides, we demonstrate the participation of Cth2 (through its TZF-domain) and Dun1 in the transcriptional induction of RNR3 under long-term iron-deficient conditions. And importantly, unlike other stresses, RNR3 is physiologically relevant when iron is scarce.