Development of an Antisense Oligonucleotide-Based Method to Manipulate RNA Editing of AMPAR Subunits Helena Anne Chaytow School of Biological Sciences Royal Holloway, University of London Research thesis submitted for the degree of Doctor of Philosophy Declaration of Authorship I, Helena Anne Chaytow, hereby declare that this thesis and the work presented in it is entirely my own. Where I have consulted the work of others, this is always clearly stated. Signed: ______________________ Date: ________________________ 2 ABSTRACT AMPA receptors (AMPARs) are a subset of ionotropic glutamate receptor composed of one or more of four subunits (GluA1-4) and are essential for normal synaptic function. The GluA2 subunit undergoes RNA editing at a specific base, converting the amino acid from glutamine to arginine, which is critical for regulating calcium permeability. RNA editing is performed by Adenosine Deaminases Acting on RNAs (ADARs). ADAR2 exists as multiple alternatively-spliced variants within mammalian cells and some have been shown to reduce their editing efficiency. RNA editing in AMPARs is inefficient in patients with Amyotrophic Lateral Sclerosis and manipulating this process could be therapeutic against AMPAR-triggered neuronal cell death. Antisense oligonucleotides (ASOs) are bases with chemically altered backbones used to manipulate DNA or RNA processing through complementary base pairing. ASOs were used to alter the GluA2 RNA editing event, either by disrupting the GluA2 double-stranded RNA structure essential for editing or by affecting the alternative splicing of ADAR2. The effects of specific ASOs on RNA editing were assessed by transfection into cell lines. Editing was quantified by an RT-PCR-based assay on RNA extracts then densitometric analysis of BbvI digestion products. ASOs targeting the secondary structure of the GluA2 subunit disrupted editing at this site to 50% of control in HeLa cells, and further disrupted editing to less than 10% in SH-SY5Y cells endogenously expressing the GluA2 subunit. ASOs targeting the ADAR2 transcript successfully inhibited the expression of a less efficient isoform, which led to increased editing of 30% compared to control HeLa cells. Further work will include building on attempts to introduce the antisense transcripts into primary cortical cultures to analyse downstream effects of reduced editing. This is the first example of increasing RNA editing using ASOs and provides a number of potential tools to investigate associated cellular processes and implications of RNA editing. 3 ACKNOWLEDGEMENTS This thesis would not have been possible without the help of several important people. Firstly, I would like to thank my supervisor Philip Chen for his endless patience and guidance throughout the past four years. I am extremely grateful for all of the support he has given me which has allowed me to grow and develop as a scientist. Thank you also to my second supervisor George Dickson, whose depth of knowledge and constructive feedback has led to some very enjoyable meetings. It was a pleasure working under the supervision of both of them. Thanks also go to Linda Popplewell for her wisdom in all things antisense, and for her most excellent advice during chapter revisions. Many thanks go to Sharifah for her instruction on techniques during the first two years of my PhD, and to Simona Ursu for being the most knowledgeable, hardworking and reliable lab technician a student could wish for. I am also incredibly grateful to the Motor Neuron Disease Association for funding this project, and so allowing me to complete this PhD. Thank you to everyone in Lab 302A, Office 404 and the Dickson lab for the company through the hours of lab work, the sarcasm, the lab-based humour and the endless cups of tea. In particular, thank you to Versha, Eva, Kate and Jade for being ever-present through the highs and lows of PhD life, I am not sure I would have finished without you there. Thank you to Annemari, Em and Bex, to Jo, Rob and Ali, for reminding me that there is life outside of science and for always being there with a glass of wine. Finally, and most importantly, I would like to thank my family for being behind me all the way through this PhD. My mother, Marysia, and sisters Alice and Susie have always had complete faith in my abilities, and it is with their love and support in mind that I submit this thesis. 4 ABBREVIATIONS LIST 25mer PMO 25 bases long 2'-F 2'-deoxy-2'-fluoro 2'-MOE 2’-O-methyoxyethyl 30mer PMO 30 bases long 5-HT 5-hydroxytryptamine; serotonin AAV adeno-associated virus ADAR adenosine deaminase acting on RNA ALS amyotrophic lateral sclerosis AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid APOBEC apolipoprotein B mRNA editing catalytic polypeptide-like enzyme family ASO antisense oligonucleotide BBP branch point binding protein bcDNA bicyclo-DNA CNS central nervous system CSF cerebrospinal fluid DIV days in vitro DMD Duchenne muscular dystrophy DPRs dipeptide repeats dsRBM double-stranded RNA binding motif E15 embryonic day 15 ECS exon complementary sequence ESEs exon splice enhancers ESSs exon splice silencers fALS familial amyotrophic lateral sclerosis FACS fluorescence-activated cell sorting F-PMO fluorescent PMO with a 5' carboxyfluorescein modification FTLD frontotemporal lobe dementia FUS fused in sarcoma protein GABA gamma-aminobutyric acid hnRNPs heterogeneous nuclear ribonucleoproteins HSF Human Splice Finder IDLV integration-deficient lentiviral vector iPSC-MNs induced pluripotent stem cell motor neurons ISEs intron splice enhancers ISSs intron splice silencers LBD ligand binding domain 5 LG-E4 plasmid lentiguide-puro plasmid containing the E4 sequence LNA locked nucleic acid L-PMO leashed PMO LTD long-term depression LTP long-term potentiation MaxExpect maximum expected accuracy miRNA microRNA MOI multiplicity of infection NMDA N-methyl-D-aspartate OMe-PS 2’-O-methylphosphorothioate P7 postnatal day 7 PBS phosphate-buffered saline PCR polymerase chain reaction PEI polyethyleneimine PESE putative exon splice enhancer PESS putative exon splice silencer PMO phosphorodiamidate morpholino oligonucleotide PNA peptide nucleic acid pri-miRNA primary microRNA Q glutamine R arginine RNA ribonucleic acid RNA-seq whole-transcriptome sequencing RT-PCR reverse transcriptase-polymerase chain reaction sALS sporadic amyotrophic lateral sclerosis SMA spinal muscular atrophy snoRNA small nucleolar RNA snRNPs small nuclear ribonucleoproteins SOD1 superoxide dismutase 1 SR proteins serine-arginine proteins TARPs transmembrane AMPA receptor regulatory proteins tcDNA tricyclo-DNA TDP-43 TAR DNA-binding protein-43 TMD transmembrane domain UTR untranslated region 6 TABLE OF CONTENTS Abstract ........................................................................................................................ 3 Acknowledgements ....................................................................................................... 4 Abbreviations List ......................................................................................................... 5 Table of Contents ......................................................................................................... 7 Table of Figures .......................................................................................................... 12 1 Literature Review ................................................................................................. 16 1.1 AMPA Receptors .......................................................................................... 16 1.1.1 AMPA Receptor Structure ..................................................................... 16 1.1.2 AMPA Receptor Function ...................................................................... 17 1.1.3 AMPA Receptor Regulation ................................................................... 18 1.1.4 Post-transcriptional Modifications .......................................................... 19 1.1.5 The GluA2 Subunit ................................................................................ 20 1.2 RNA Editing .................................................................................................. 23 1.2.1 Forms of RNA Editing ............................................................................ 23 1.2.2 ADAR Structure ..................................................................................... 26 1.2.3 RNA Editing of Non-coding RNAs .......................................................... 28 1.2.4 Identification of RNA Editing Sites ......................................................... 30 1.2.5 ADAR2 .................................................................................................. 31 1.2.6 Alternative Splicing of ADAR2 ............................................................... 31 1.2.7 RNA Editing in the Central Nervous System .......................................... 34 1.2.8 RNA Editing in Glutamate Receptors ..................................................... 36 1.3 RNA editing at the Q/R Site of GluA2 ........................................................... 37 1.3.1 Discovery of the Exon Complementary Sequence and Imperfect Repeat 37 1.3.2 Q/R Site Editing and Disease ................................................................ 38 1.3.3 Transgenic Models Changing the Q/R Site ............................................ 39 1.4 Amyotrophic Lateral Sclerosis ...................................................................... 41 1.4.1 Incidence and Symptoms .....................................................................