UPSTREAM OPEN READING FRAMES DIFFERENTIALLY REGULATE GENE- SPECIFIC TRANSLATION IN THE INTEGRATED STRESS RESPONSE Sara Kathryn Young Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology Indiana University July 2016 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ____________________________________ Ronald C. Wek, Ph.D., Chair ____________________________________ Murray Korc, M.D. Doctoral Committee ____________________________________ Amber L. Mosley, Ph.D. May 13, 2016 ____________________________________ John J. Turchi, Ph.D. ii © 2016 Sara Kathryn Young iii ACKNOWLEDGEMENTS I am foremost indebted to Dr. Ronald C. Wek for being an extraordinary mentor who has worked to mold me into a capable research scientist. I appreciate the time and dedication of my research committee: Dr. Murray Korc, Dr. Amber L. Mosley, and Dr. John J. Turchi. Past and current members of the Wek lab were paramount to the work presented in this thesis, particularly Dr. Thomas D. Baird with his invaluable contribution to the EPRS project and overall support. I would also like to the thank the faculty and staff of the Department of Biochemistry for their generous support and guidance. This work was supported by National Institutes of Health Grant GM049164 to R.C.W. iv Sara Kathryn Young UPSTREAM OPEN READING FRAMES DIFFERENTIALLY REGULATE GENE- SPECIFIC TRANSLATION IN THE INTEGRATED STRESS RESPONSE Gene expression is a highly coordinated process that relies upon appropriate regulation of translation for protein homeostasis. Regulation of protein synthesis largely occurs at the initiation step in which the translational start site is selected by ribosomes and associated initiating factors. In addition to the coding sequences (CDS) for protein products, short upstream open reading frames (uORFs) located in the 5’-leader of mRNAs are selected for translation initiation. While uORFs are largely considered to be inhibitory to translation at the downstream CDS, uORFs can also promote initiation of CDS translation in response to environmental stresses. Multiple transcripts associated with stress adaptation are preferentially translated through uORF-mediated mechanisms during activation of the Integrated Stress Response (ISR). In the ISR, phosphorylation of α subunit of the translation initiation factor eIF2α (eIF2α~P) during environmental stresses results in a global reduction in protein synthesis that functions to conserve energy and nutrient resources and facilitate reprogramming of gene expression. Many key regulators of the ISR network are subject to preferential translation in the response to eIF2α-P. These preferentially translated genes include the pro-apoptotic transcriptional activator Chop that modifies gene expression programs, feedback regulator Gadd34 that targets the catalytic subunit of protein phosphatase 1 to dephosphorylate eIF2α~P, and glutamyl-prolyl tRNA synthetase Eprs that increases the charged tRNA pool and primes the cell for resumption of protein synthesis after stress remediation. Ribosome bypass of at least one inhibitory uORF is a common theme between Chop, Gadd34, and Eprs, which allows for their regulated expression in response to cellular stress. However, different features encoded within the uORFs of v the Chop, Gadd34, and Eprs mRNAs provide for regulation of their inhibitory functions, illustrating the complexities of uORF-mediated regulation of gene-specific translation. Importantly, preferentially translated ISR targets can also be transcriptionally regulated in response to cellular stress and misregulation of transcriptional or translational expression of Gadd34 can elicit maladaptive cell responses that contribute to disease. These mechanisms of translation control are conserved throughout species, emphasizing the importance of translation control in appropriate gene expression and the maintenance of protein homeostasis and health in diverse cellular conditions. Ronald C. Wek, Ph.D., Chair vi TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................... x ABBREVIATIONS .......................................................................................................... xiii CHAPTER 1. INTRODUCTION ....................................................................................... 1 1.1 Regulation of gene expression by uORFs .................................................................. 1 1.2 uORFs and their function in the regulation of downstream translation initiation ......... 5 1.3 Translation regulation during the Integrated Stress Response ................................... 7 1.4 Preferentially translated mRNAs play diverse roles in response to cellular stress ..... 8 1.5 Ribosome reinitiation in the ISR ................................................................................ 12 1.6 Ribosome bypass in the ISR ..................................................................................... 15 1.7 Cross-regulation between the ISR and other cellular stress pathways ..................... 18 CHAPTER 2. EXPERIMENTAL PROCEDURES .......................................................... 21 2.1 Cell culture and generation of stable cell lines .......................................................... 21 2.2 Mice .......................................................................................................................... 22 2.3 Immunoblot analyses ................................................................................................ 22 2.4 mRNA measurements by qPCR ............................................................................... 23 2.5 Total RNA and DNA measurements ......................................................................... 25 2.6 Polysome profiling and sucrose gradient ultracentrifugation .................................... 25 2.7 Plasmid constructions and luciferase assays ........................................................... 25 2.8 In vitro transcription and translation assays .............................................................. 30 2.9 Cell number and viability assays ............................................................................... 31 2.10 Statistical analyses ................................................................................................. 32 CHAPTER 3. RESULTS: RIBOSOME REINITIATION DIRECTS GENE-SPECIFIC TRANSLATION AND REGULATES THE INTEGRATED STRESS RESPONSE ......... 33 3.1 eIF2α~P is required for Gadd34 transcription and translation, but Crep expression occurs independent of eIF2α~P .................................................................. 33 vii 3.2 Preferential translation of Gadd34 features an inhibitory uORF ............................... 37 3.3 Gadd34 translation control involves bypass of an inhibitory uORF .......................... 41 3.4 The inhibitory function of Gadd34 uORF2 is reliant on Pro-Pro-Gly juxtaposed to the uORF2 stop codon ................................................................................................ 42 3.5 Crep translation is dampened by an inhibitory uORF in an eIF2α~P independent manner ....................................................................................................... 50 3.6 Regulatory properties of Gadd34 uORF2 are transferable to a heterologous 5’-leader derived from Crep ............................................................................................ 54 3.7 Alterations in the regulatory features of Gadd34 uORF2 affect cell viability during ER stress ............................................................................................................. 58 CHAPTER 4. RESULTS: RIBOSOME ELONGATION STALL DIRECTS GENE- SPECIFIC TRANSLATION CONTROL IN THE INTEGRATED STRESS RESPONSE .................................................................................................................... 63 4.1 The inhibitory function of Chop uORF is reliant upon an Ile-Phe-Ile sequence ........ 63 4.2 Translation of an Ile-Phe-Ile sequence in the Chop uORF results in an elongation stall .................................................................................................................................. 73 4.3 Alterations in Chop uORF translation control change the dynamics of CHOP expression ....................................................................................................................... 79 4.4 Alterations in Chop uORF translation control affect cell viability ............................... 85 CHAPTER 5. RESULTS: TRANSLATION REGULATION OF THE GLUTAMYL- PROLYL TRNA SYNTHETASE GENE EPRS THROUGH BYPASS OF UORFS WITH NON-CANONICAL INITIATION CODONS .................................................................... 88 5.1 Eprs expression is increased in response to eIF2α~P through enhanced translation ........................................................................................................................................ 88 5.2 Preferential translation of Eprs features two uORFs with non-canonical initiation codons ............................................................................................................................ 89 viii 5.3 Eprs translational control involves bypass of two uORFs with non-canonical initiation codons ...........................................................................................................................