Post-transcriptional Regulation of Gene Expression in Response to Iron Deficiency in Saccharomyces cerevisiae by Sandra Viviana Vergara Program in Genetics and Genomics Duke University Date:_______________________ Approved: ___________________________ Dennis J. Thiele, Ph.D., Supervisor ___________________________ Matthias Gromeier, M.D. ___________________________ Jack Keene, Ph.D. ___________________________ Daniel Lew, Ph.D. ___________________________ William Marzluff, Ph.D. Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program in Genetics and Genomics of Duke University 2010 ABSTRACT Post-transcriptional Regulation of Gene Expression in Response to Iron Deficiency in Saccharomyces cerevisiae by Sandra Viviana Vergara Program in Genetics and Genomics Duke University Date:_______________________ Approved: ___________________________ Dennis J. Thiele, Ph.D., Supervisor ___________________________ Matthias Gromeier, M.D. ___________________________ Jack Keene, Ph.D. ___________________________ Daniel Lew, Ph.D. ___________________________ William Marzluff, Ph.D. An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program in Genetics and Genomics in the Graduate School of Duke University 2010 Copyright by Sandra Viviana Vergara 2010 Abstract The ability of iron (Fe) to easily transition between two valence states makes it a preferred co-factor for innumerable biochemical reactions, ranging from cellular energy production, to oxygen transport, to DNA synthesis and chromatin modification. While Fe is highly abundant on the crust of the earth, its insolubility at neutral pH limits its bioavailability. As a consequence, organisms have evolved sophisticated mechanisms of adaptation to conditions of scarce Fe availability. Studies in the baker’s yeast Saccharomyces cerevisiae have shed light into the cellular mechanisms by which cells respond to limited Fe-availability. In response to Fe- deficiency, the transcription factors Aft1 and Aft2 activate a group of genes collectively known as the Fe-regulon. Genes in this group encode proteins involved in high-affinity plasma membrane Fe-transport and siderophore uptake, as well as Fe-mobilization from intracellular stores and heme re-utilization. Concomitant with the up-regulation of the Fe-regulon, a large number of mRNAs encoding Fe-dependent proteins as well as proteins involved in many Fe-dependent processes are markedly down regulated. Thus, in response to low Fe-levels the cell activates the Fe-uptake and mobilization systems, while down-regulating mRNAs involved in highly Fe-demanding processes leading to a genome-wide remodeling of cellular metabolism that permits the funneling of the limiting Fe to essential Fe-dependent reactions. iv The Fe-regulon member Cth2 belongs to a family of mRNA-binding proteins characterized by an RNA-binding motif consisting of two tandem zinc-fingers of the CX 8CX 5CX 3H type. Members of this family recognize and bind specific AU-rich elements (AREs) located in the 3’untranslated region (3’UTRs) of select groups of mRNAs, thereby promoting their rapid degradation. In response to Fe-limitation, Cth2 binds ARE sequences within the 3’UTRs of many mRNAs encoding proteins involved in Fe- homeostasis and Fe-dependent processes, thereby accelerating their rate of decay. Work described in this dissertation demonstrates that the Cth2 homolog , Cth1, is a bona fide member of the Fe-regulon, binds ARE-sequences within the 3’UTRs of select mRNAs and promotes their decay. Cth1 and Cth2 appear to be only partially redundant; Cth1 preferentially targets mRNAs encoding mitochondrial proteins, while Cth2 promotes the degradation of most of Cth1 targets in addition to other mitochondrial and non-mitochondrial Fe-requiring processes. The coordinated activity of Cth1 and Cth2 results in dramatic changes in glucose metabolism. In addition, experiments described in this dissertation indicate that the CTH1 and CTH2 transcripts are themselves subject to ARE-mediated regulation by the Cth1 and Cth2 proteins, creating an auto- and trans- regulatory circuit responsible for differences in their expression. Finally, work described here demonstrates that Cth2 is a nucleocytoplasmic shuttling protein and that shuttling is important for the early determination of cytosolic mRNA-fate. v Contents Abstract ......................................................................................................................................... iv List of Tables .................................................................................................................................. x List of Figures ............................................................................................................................... xi Acknowledgements .................................................................................................................. xiii 1. Introduction ............................................................................................................................... 1 1.1. Co-evolution of life and transition metal bioavailability ........................................... 1 1.2. Iron in biology – essential, harmful, and hard to get ................................................. 3 1.2.1. Iron-Sulfur clusters proteins ..................................................................................... 4 1.2.1. Heme proteins ............................................................................................................. 5 1.2.3. Other Fe-coordinating proteins ................................................................................ 6 1.3. Iron Homeostasis in bacteria ....................................................................................... 10 1.3.1. Iron uptake and storage ........................................................................................... 10 1.3.2 The ferric uptake repressor, Fur .............................................................................. 12 1.3.3 RyhB-mediated response to iron deficiency .......................................................... 13 1.4. Iron homeostasis in baker’s yeast ................................................................................ 14 1.4.1. Iron uptake and storage ........................................................................................... 14 1.4.2. Transcriptional regulation of iron homeostasis ................................................... 17 1.4.3. Post-transcriptional regulation of iron homeostasis ........................................... 20 1.4.4. Metabolic regulation of iron homeostasis ............................................................. 24 1.5. Iron homeostasis in mammals ..................................................................................... 26 1.5.1. Iron uptake and storage ........................................................................................... 26 vi 1.5.2. Systemic regulation of iron balance – hepcidin ................................................... 30 1.5.3. Cellular regulation of iron balance – the IRE/IRP system .................................. 31 1.5.4. Disorders of iron of balance .................................................................................... 32 1.6. Post-transcriptional regulation of mRNA stability by ARE-binding proteins ..... 35 1.6.1. Molecular mechanisms of mRNA decay ............................................................... 35 1.6.2. AU-rich elements (AREs) ........................................................................................ 39 1.6.3. ARE-binding proteins (AUBPs) .............................................................................. 40 1.6.3.1. CX 8CX 5CX 3H-proteins in mammals .................................................................... 42 1.6.4. ARE-mediated mRNA decay – AMD .................................................................... 46 1.7. CX 8CX 5CX 3H proteins in yeast – Cth1 and Cth2 ....................................................... 48 2. Cooperation of two mRNA-binding proteins drives metabolic adaptation to iron deficiency ..................................................................................................................................... 52 2.1 Introduction ..................................................................................................................... 52 2.2 Materials and Methods .................................................................................................. 55 2.3 Results .............................................................................................................................. 60 2.3.1 Cth1 function in response to Fe-deficiency ............................................................ 60 2.3.2 Aft1 and Aft2 regulate CTH1 transcription ........................................................... 63 2.3.3 Cth1 stimulates mRNA turnover ............................................................................ 66 2.3.4 Cth1 targets mRNAs mostly encoding mitochondrial proteins ......................... 70 2.3.5 Cth1/Cth2-dependent changes in carbohydrate metabolism .............................. 86 2.3.6 Iron deficiency elicits changes in glucose metabolism ......................................... 92 2.4 Discussion .......................................................................................................................