University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES Centre for Biological Sciences Control of translation initiation and neuronal subcellular localisation of mRNAs by G-quadruplex structures by James Philip Robertson Schofield Thesis for the degree of Doctor of Philosophy 2015 i UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES CENTRE FOR BIOLOGICAL SCIENCES Doctor of Philosophy Control of translation initiation and neuronal subcellular localisation of mRNAs by G- quadruplex structures by James Philip Robertson Schofield The translation of mRNA is a key regulatory step in control of gene expression. The primary sequence of an mRNA determines much of the regulation of translation, including the location of translation initiation. The efficiency of translation initiation is determined by the initiation codon and its context. Translation initiation occurs at the canonical initiation codon, AUG, or a less efficient near canonical non-AUG alternative initiation codon (AIC). The secondary structure of an mRNA impacts its translation, principally by inhibiting binding and/or migration of ribosomal subunits. Hairpins form by hydrogen bonding between Watson-Crick complementary base-pairs. Guanine-rich nucleotide sequences form planar tetrads via hydrogen bonds and stack as stable G-quadruplex structures. Much has been reported on the impact of hairpins in regulating translation and G-quadruplex structures are emerging as an important factor in the regulation of translation of some mRNAs. Two-pore potassium leak (K2P) channels set and maintain the resting membrane potential of cells, so precise regulation of K2P protein expression in cells is critical to cell behaviour and survival. The 5’ untranslated regions (5’ UTR) of K2P channels are poorly annotated in databases. This thesis details investigations of 5’ UTR primary sequence and secondary structure effects on the expression of K2P leak channels. We characterised an extension to the annotated 5’ UTR sequence of Task3 by 5’RACE. The extension is characterised by a 5’ terminal (GGN) repeat which we show forms a G-quadruplex structure and inhibits translation of Task 3. G-quadruplex formation was measured by circular dichroism and increased fluorescence of Mesoporphyrin IX dihydrochloride. RT-PCR of Task3-FLAG mRNA relative to β-actin mRNA in polysome fractions of transfected HeLa cells suggests the inhibition of Task3 protein synthesis results from an inhibition of translation initiation due to the 5’-terminal G-quadruplex. Results suggest the inhibition of Task3 protein synthesis can be modulated by RNA-binding proteins. hnRNPA2 is shown to relieve translational inhibition, and the DEAH/RHA RNA helicases, DHX29, DHX30 and DHX36 differentially regulate translation from Task3 mRNA dependent on the presence of the 5’ terminal G- quadruplex. We also investigate the use of small ligands TMPyP4 and Hippuristanol in modulating expression of Task3 in a G-quadruplex-dependent manner. Task3 is expressed in neuronal cells and is predicted to be able to exert control over local membrane potential. The local translation of mRNAs has been demonstrated in a variety of cells. In neurons, local translation of some mRNAs at synapses have been shown in plastic changes. G-quadruplex structures have been shown to affect mRNA subcellular targeting and direct protein synthesis at the synapse. We describe detection of Task3-GFP mRNA by Fluorescence In Situ Hybridisation (FISH) in transfected primary cortical neurons. It was found for the first time that the 5’-terminal G-quadruplex in Task3 mRNA appears to mediate localisation of Task3-GFP mRNA to discrete neurite particles. The roles of G- quadruplex-interacting RNA binding proteins (RBPs), hnRNP A2 and Purα, in the trafficking of Task3 mRNA was investigated, however, the mechanism of neurite delivery of Task3 ii mRNA requires further research. Dysregulation of this mechanism would cause perturbations to individual synapse excitability and therefore contribute to neuronal behaviour. FXR2 is a paralog of the neuronal translation regulator RBP, Fragile X Mental Retardation Protein (FMRP). Ribosome profiling of mouse embryonic stem cells identified Fxr2 to be subject to alternative upstream translation initiation. Results here demonstrate the production of different N-terminal length isoforms of Fxr2 from alternative translation initiation, primarily from GUG codon, -219 from the annotated AUG initiation codon. Investigation of the 5’ UTR of Fxr2 revealed a high concentration of guanine residues. We show evidence supporting G-quadruplex structures within its 5’UTR. This is the first report of potential G-quadruplex mediated control of AIC usage in an mRNA. iii Contents Declaration of authorship ............................................................................................... xi Acknowledgements ....................................................................................................... xii Abbreviations ............................................................................................................... xiii List of figures................................................................................................................ xiv List of Tables ............................................................................................................... xvii Chapter One ................................................................................................................... 1 Introduction ................................................................................................................... 1 1.1 Regulation of translation initiation ........................................................................ 1 1.1.1 mRNA primary sequence determines initiation codon usage ....................... 5 1.1.2 Leaky scanning creates diverse translation products .................................... 8 1.2 mRNA secondary structures regulate initiation codon usage ................................ 12 1.2.1 Hairpins impact translational mechanisms .............................................. 12 1.2.2 G-quadruplexes impact translational mechanisms .................................. 14 1.3 RNA-binding proteins and the 5’ UTRs of mRNAs ..................................................... 21 1.3.1 RNA Helicases resolve secondary structure ................................................. 21 1.4 Modulating RNA G-quadruplex effects with small-molecules ................................... 27 1.5 Subcellular localisation of mRNA ......................................................................... 28 1.5.1 mRNA structural motifs determine mRNA targeting ................................... 32 1.6 FMRP is a G-quadruplex binding RBP and its mRNA contains G-quadruplexes ....... 33 1.7 Local translation affects synaptic plasticity .......................................................... 35 1.7.1 G-quadruplex mediated delivery of mRNA to synaptic spines for controlled local protein synthesis ........................................................................................................ 40 1.7.2 G-quadruplex expansion diseases deregulate processing of G-quadruplex mRNA .................................................................................................................................. 47 1.8 Two-pore potassium leak channels .......................................................................... 47 1.8.1 Potassium leak channels set the baseline membrane potential of neurons48 1.9 Regulated expression of K2P channel proteins .......................................................... 52 iv 1.10 Alternative translation initiation in K2P channel, Trek1 ............................................ 54 1.11 Bioinformatic screen of mRNAs identified an evolutionarily conserved sequence in the 5’ UTR of K2P channel, Traak ........................................................................... 55 1.12 Alternative translation initiation in Fxr2 ................................................................. 56 1.13 Aims ...................................................................................................................... 59 Chapter Two ................................................................................................................. 60 Materials and Methods ................................................................................................. 60 Chapter 2: Materials and Methods ................................................................................ 60 2.1 Measurement of RNA/DNA concentration................................................................ 60 2.2 PCR