CPEB4 function in macrophages Clara Suñer Navarro Aquesta tesi doctoral està subjecta a la llicència Reconeixement 4.0. Espanya de Creative Commons. Esta tesis doctoral está sujeta a la licencia Reconocimiento 4.0. España de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution 4.0. Spain License. Universitat de Barcelona Facultat de Farmàcia i Ciències de l’Alimentació IRB Barcelona Programa de Doctorat en Biomedicina CPEB4 function in macrophages Aquesta tesi ha estat realitzada per la Clara Suñer Navarro sota la direcció del Dr. Raúl Méndez de la Iglesia, a l’Institut de Recerca Biomèdica de Barcelona (IRB Barcelona). Es presenta aquesta memòria per optar al títol de doctora per la Universitat de Barcelona en el Programa de Doctorat en Biomedicina. Raúl Méndez de la Iglesia Antonio Zorzano Olarte Clara Suñer Navarro Director de tesi Tutor de tesi Doctoranda Barcelona 2018 Para los cuatro. “Not only to be receiver, but also to be provider” Buddhist proverb Preface What is the meaning of life? Throughout history, great efforts have been made trying to understand which is the significance of living or existence. Philosophical, scientific, theological and metaphysical approaches have proposed multiple answers to questions such as “Why are we here?”, “What is life all about?”, or “What is the purpose of existence?”. Scientific contributions have focused primarily on describing empirical facts about the universe, exploring the context and parameters concerning what life is and aiming to describe how it works. Though life definition is still a challenge, from the biologist point of view lifeforms are self-organizing systems that regulate their internal environments as to maintain this organized state, also known as homeostasis, and reproduce to continue life over multiple generations. Importantly, homeostasis maintenance needs the capacity to sense and adapt to the external environment. Throughout this thesis, we will focus on a specific subset of mechanisms developed to respond to a certain stimulus. These mechanisms, if successful, return life to its homeostatic state but, upon failure, may lead to life- threating conditions. I am afraid we will not provide a new answer to the question “What is the meaning of life?”, however we will further describe its complexity and fragility and, one could extrapolate from that, how precious life is. 9 INDEX Index PREFACE ____________________________________________ 9 ABBREVIATIONS ____________________________________ 21 ABSTRACT ___________________________________________ 27 INTRODUCTION ____________________________________ 31 1. Regulation of protein synthesis ......................................................... 33 1.1. pre-mRNA processing ......................................................................................34 1.2. Mature mRNA structure ..................................................................................37 1.3. mRNA translation .............................................................................................38 1.3.1. Translation initiation .................................................................................38 1.3.1.1. Regulation of translation initiation ................................................40 1.3.2. Translation elongation ..............................................................................42 1.3.3. Translation termination and ribosome recycling .................................43 1.4. mRNA localization ............................................................................................44 1.5. mRNA decay ......................................................................................................46 1.5.1. Basal mRNA decay ..............................................................................46 1.5.2. Quality control (QC) decay ................................................................46 1.6. mRNA deadenylation .......................................................................................47 1.7. Cytoplasmic Polyadenylation ...........................................................................49 1.7.1. Cytoplasmic Polyadenylation Element Binding Proteins ...................50 1.7.2. Cytoplasmic Polyadenylation Elements (CPEs) ...................................51 1.7.3. Regulation of CPEBs activity .................................................................52 1.7.4. CPEBs biological function ......................................................................54 1.7.5. Mechanisms regulating CPEBs expression ...........................................56 2. The Innate immune system ............................................................... 57 2.1. The mononuclear phagocytic system (MPS) .................................................59 2.2. Macrophage function during an inflammatory response ............................60 2.3. Macrophage activation by LPS ........................................................................61 13 2.4. TLR4 signalling pathway ..................................................................................62 BOX 1. MAPK signalling cascades ....................................................................63 2.4.1. MyD88-dependent signal transduction .................................................64 2.4.2. TRIF-dependent signal transduction .....................................................64 2.4.3. Role of MAPK in LPS response: p38, ERK, JNK .............................65 2.4.4. NF- κB .......................................................................................................66 2.4.5. Autocrine loops in LPS response ...........................................................67 2.5. Inflammation resolution: TLR4 and NF-κB negative Regulators .............68 2.6. Post-transcriptional control during the LPS response .................................71 2.6.1. mRNA decay during the LPS response .................................................71 2.6.2. Translational regulation during the LPS response ...............................74 2.6.3. CPEBs in LPS response ...........................................................................75 OBJECTIVES _________________________________________ 77 RESULTS _____________________________________________ 81 1. CPEB4 is upregulated upon LPS stimulation in Macrophages .....................83 2. CPEB4 mRNA stability is regulated during the LPS response via p38a-HuR-TTP .....................................................................................................85 3. CPEB4 is phosphorylated during the LPS response ......................................90 4. Defining CPEB4 function during the late LPS response in BMDMs.......... 92 4.1. CPEB4 regulates negative feedback inhibitors of the LPS response ..92 BOX 2. Revisiting CPEs definition ...........................................................94 4.2. Assessing CPEB4 function in BMDMs by ribosome profiling ............97 4.3. Distinct behaviours of CPEB4 targets in Cpeb4KO BMDMs ..............103 4.4. Interplay between CPEB4 and TTP during the LPS response ............107 4.4.1. Key genes of the inflammatory response are regulated by TTP and CPEB4 ............................................................................................108 4.4.2. An ARE/CPE score predicts mRNA behaviour during the LPS response .................................................................................................112 14 4.5. Transcriptional control of the LPS response in Cpeb4KO BMDMs ...116 5. CPEB4 function in quiescent BMDMs .............................................................118 6. CPEB4 function in macrophages in vivo .........................................................122 6.1. Myeloid-specificCpeb4 KO mice in homeostasis .....................................124 6.2. Decreased survival of Cpeb4MKO mice to LPS-induced homeostatic shock ......................................................................................................................127 DISCUSSION _________________________________________ 133 1. Inflammation resolution requires CPEB4-mediated translational control ....................................................................................................................135 2. CPEB4, a new player in LPS response ..............................................................136 3. The MAPKs p38 and ERK1/2 regulate CPEB4 levels and activity during the LPS response ......................................................................................137 α 4. Relevance of CPEB4 hyperphosphorylation for LPS response ...................139 5. Closing the circuit: CPEB4 targets are enriched in MAPK pathway-related genes ..........................................................................................140 6. CPEB4 promotes inflammation resolution during the LPS response ..................................................................................................................................142 7. AREs and CPEs define the kinetics of mRNAs during the LPS response .................................................................................................................143 7.1. HuR, TTP and CPEB4 define different mRNA expression waves during the LPS response .............................................................................143 7.2. CPEB4 polyadenylating function stabilizes CPE-containing mRNAs during the LPS response .............................................................145 7.3. The Combinatorial