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The Role of Pumilio 2 in Axonal Outgrowth by Dani Sarkis a Thesis The Role of Pumilio 2 in Axonal Outgrowth by Dani Sarkis A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto Copyright c 2012 by Dani Sarkis Abstract The Role of Pumilio 2 in Axonal Outgrowth Dani Sarkis Master of Science Graduate Department of Physiology University of Toronto 2012 Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and transla- tional regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant nega- tive mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length com- pared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth. ii Dedication To Ahed Khouri and Farid Sarkis, my mother and father. For everything you have done for me. For your unconditional love and support. To Gabi Sarkis, my brother, my best friend, and idol. iii Acknowledgements First and foremost, I would like to thank my supervisor, Dr. Philippe Monnier, for giving me the chance to work on this project. I would also like to thank my advisory committee members, Dr. Zhong-Ping Feng and Dr. James Eubanks, for their insight and for ensuring I was on the right track towards obtaining my MSc. I would also like to acknowledge the help and guidance of our research associate, Dr. Nardos Tassew, PhD. Dr. Tassew taught me the skills that I needed to complete this project, and had very enlightening discussions with me to help me better understand my project, and I am grateful for her patience and help. Thank you Dr. Tassew and Dr. Paromita Banerjee for your help with editing this thesis. I must also thank my friends Gemma Higgs, MSc for help with editing this thesis, Dr. Anne Wheeler, PhD for her helpful feedback and getting me into research in the first place, and Elena Sidorova, MSc for her continuous support and encouragement. This project was funded by two scholarships from the Vision Science Research Pro- gram (VSRP) at the Toronto Western Research Institute. iv Contents 1 Introduction 1 1.1 mRNA Translation . 1 1.1.1 Overview of mRNA Translation in Eukaryotes . 1 1.1.2 Local Translation, RNA transport, and mRNA Binding Proteins . 2 1.2 Axonal Outgrowth . 4 1.3 The Embryonic Chicken Dorsal Root Ganglia as a Model for Axonal Out- growth . 6 1.4 Pumilio - a Founding Member of the Puf Family . 7 1.5 Pumilio is Involved in Embryonic and Germline Development and Cell Cycle Regulation . 9 1.6 Pumilio 1 and Pumilio 2: Vertebrate Homologues of the Drosophila Pumilio 10 1.7 Pumilio 2 and the Eukaryotic Initiation Factor 4E (eIF4E) . 12 1.8 Pumilio in the Nervous System . 13 1.9 Study Rationale . 14 1.10 Hypothesis and Aims . 17 2 Materials and Methods 18 2.1 Cloning . 18 2.1.1 Cloning of dominant negative PUM2 (dnPUM2) . 18 2.1.2 Cloning of PUM2W349G-EYFP in pEYFP-N1 . 19 v 2.1.3 Cloning of PUM2 microRNA (PUM2miRNA) . 20 2.1.4 Cloning of chicken PUM2 in pcDNA3.1 (-)/myc-His A . 21 2.1.5 Cloning of eIF4E-T2A-PUM2EYFP . 22 2.2 Cell Culture . 23 2.2.1 Transfection of Cell Lines . 24 2.3 Chicken Embryos . 24 2.4 Dorsal Root Ganglia (DRG) Neurons . 25 2.4.1 Dissection . 25 2.4.2 Nucleofection . 25 2.5 Retinal Flat Mounts . 26 2.6 Virus Preparation . 26 2.7 Fiber Tracing . 27 2.8 In Ovo Electroporation . 27 2.9 Immuno-cytochemistry . 29 2.10 Western Blots . 29 2.11 Microscopy . 30 2.12 Live Imaging . 31 2.13 Statistical Analysis . 31 3 Results 32 3.1 Construct Verification . 32 3.2 PUM2 is transported along the axons in DRG neurons and RGCs . 32 3.3 PUM2 overexpression results in shorter axons in dissociated DRG neurons 33 3.4 Expression of dnPUM2 or PUM2W349G results in shorter axons in disso- ciated DRG neurons . 33 3.5 PUM2 silencing . 34 3.5.1 PUM2 silencing hinders axonal outgrowth in dissociated DRG neu- rons . 34 vi 3.5.2 PUM2 silencing does not affect eye size at E4 . 35 3.6 eIF4E fails to rescue the short axon phenotype . 35 4 Discussion 54 4.1 PUM2 localization in the growth cone and axons of RGCs and DRG neurons 55 4.2 Impaired axonal outgrowth in DRG neurons overexpressing PUM2 . 56 4.3 Impaired axonal outgrowth in DRG neurons expressing PUM2 mutants . 58 4.4 PUM2 silencing interferes with axonal outgrowth in DRG neurons . 60 4.5 eIF4E coexpression fails to rescue short axon phenotype . 61 4.6 Future Directions . 63 4.6.1 Are PUM2/Nos and PUM2/mRNA interactions necessary for nor- mal axonal outgrowth? . 63 4.6.2 Can the short axon phenotype be rescued? . 63 4.6.3 What is the cause of the short axon phenotype? . 65 4.6.4 Testing the role of PUM2 in axonal outgrowth and guidance in vivo 66 4.7 Conclusion . 67 References 67 vii List of Figures 1.1 In Situ hybridization showing the expression of Pum2 transcript in E9 eye 15 1.2 PUM2 Expression in Axons and Growth Cones of Retinal Ganglion Cells 16 2.1 Schematic Representation of eIF4E-T2A-PUM2EYFP in pT2K . 23 2.2 In Ovo Electroporation of E1.5 Embryos . 28 3.1 Western blot verifying the provided and cloned constructs . 37 3.2 Western blot verifying PUM2W349G-EYFP . 38 3.3 PUM2 is transported along the axon of RGCs . 38 3.4 PUM2 is endogenously expressed in DRG neurons . 39 3.5 PUM2 Overexpression Results in Short Axons . 40 3.6 Quantification of Axon Length in PUM2 Overexpression . 41 3.7 DRG neurons expressing dnPUM2 have shorter axons . 42 3.8 Quantification of Axonal Length in dnPUM2 Overexpression . 43 3.9 DRG neurons expressing PUM2W349G have short axons . 44 3.10 Quantification of Axonal Length in PUM2W349G Overexpression . 45 3.11 PUM2 Silencing with miRNA constructs . 46 3.12 PUM2miRNA transfected neurons had shorter axons . 47 3.13 Quantification of Axonal Length After PUM2 Silencing . 48 3.14 PUM2 Silencing and Eye Size . 49 3.15 Quantification of the effects of PUM2 silencing on eye size at E4 . 50 viii 3.16 Western Blot Confirming Coexpression of eIF4E and PUM2EYFP . 51 3.17 DRG neurons coexpressing eIF4E and PUM2 have short axons . 52 3.18 Quantification of Axonal Length in eIF4E and PUM2 Coexpression . 53 4.1 Known interactions of PUM2 and possible involvement in axonal outgrowth 64 4.2 Time Lapse Imaging of DRG Neurons . 68 ix Chapter 1 Introduction 1.1 mRNA Translation 1.1.1 Overview of mRNA Translation in Eukaryotes Messenger RNA (mRNA) translation is a stage of protein biosynthesis, where mRNA is used as a template for the assembly of amino acids to produce a polypeptide chain that undergoes modifications to form the protein. Translation is a complex and intricate process that involves many factors which are required to work in concert with one an- other. Briefly, eukaryotic initiation factors (eIFs) activate the mRNA in preparation for ribosomal binding. eIFs bind to the m7G cap and the poly-A tail at the 5' and 3' ends of the mRNA, respectively. Next, the small ribosomal subunit (40S), loaded with the methionyl tRNA specialized for initiation (Met-tRNAi) and the initiation factors 1, 1A, 2, 3, and 5 must come together to form a pre-initiation complex (PIC). PIC interacts with the m7G binding eIF4E as well as eIF4G. The PIC then scans the 5' untranslated region (UTR) of the mRNA for the start codon AUG (Sonenberg and Hinnebusch, 2009). Furthermore, poly(A) binding protein (PABP) binds the poly(A) tail at the 3' end of the mRNA and binds several factors, including the termination factor eRF-3. Direct interaction between PABP and eIF4G allows for the circularization of mRNA and the 1 Chapter 1. Introduction 2 formation of a closed loop complex (Sonenberg and Dever, 2003). The GTP (Guanosine Triphosphate) on eIF2 is hydrolyzed to GDP (Guanosine Diphosphate), enabling the 40S ribosomal subunit to bind with a larger (60S) ribosomal subunit, forming an 80S ribosome necessary for translation of the mRNA (de Moor et al., 2005). Other factors bind the 3' UTR and contribute to the translation of mRNA. One such factor is the cytoplasmic polyadenylation element binding protein (CPEB). CPEB recognizes the cytoplasmic polyadenylation element (CPE) in the 3' UTR of the mRNA and improves the recruitment of cleavage and polyadenylation specificity factor (CPSF), allowing for polyadenylation of the mRNA by poly(A) polymerase, thus increasing the stability of the mRNA (Radford et al., 2008). As one would expect, mRNA translation has the potential to be regulated in many ways and at different steps as there are many factors that are involved. For example, phosphorylation of CPEB by Aurora A kinase on serine 174 is required in order for CPEB to recruit CPSF to the poly(A) signal. Another example is the suppression of cyclin B1 translation by Maskin, which can bind CPEB and eIF4E.
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