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This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • 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 author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Expression and subcellular localisation of poly(A)-binding proteins Hannah Burgess PhD The University of Edinburgh 2010 Abstract Poly(A)-binding proteins (PABPs) are important regulators of mRNA translation and stability. In mammals four cytoplasmic PABPs with a similar domain structure have been described - PABP1, tPABP, PABP4 and ePABP. The vast majority of research on PABP mechanism, function and sub-cellular localisation is however limited to PABP1 and little published work has explored the expression of PABP proteins. Here, I examine the tissue distribution of PABP1 and PABP4 in mouse and show that both proteins differ markedly in their expression at both the tissue and cellular level, contradicting the widespread perception that PABP1 is ubiquitously expressed. PABP4 is shown to be widely expressed though with an expression pattern distinct from PABP1, and thus may have a biological function in many tissues. I have shown that PABP4 associates with polysomes and described interactions between PABP4 and proteins important for PABP1-mediated translational activation suggesting that PABP4 plays a similar role in translational regulation. Translational inhibition is a common response to cell stress. Following certain cellular stresses e.g. arsenite, PABP1 localises to cytoplasmic stress granules, sites of mRNA storage. Here, I show that PABP4 is also a component of stress granules and have investigated the role of PABPs in stress granule formation. In contrast, I have shown that both PABP1 and PABP4 relocalise to the nucleus following UV irradiation in both human and mouse cell lines. In order to understand the how the UV-induced change in PABP localisation may be regulated, I have investigated the regulation of PABP nuclear export. I have found that accumulation of PABP proteins in the nucleus after UV coincides with an accumulation of poly(A)+ RNA, indicative of a block in mRNA export, an effect which has not been previously characterized. My working model is that nuclear export of PABPs may be dependent on mRNA export, inhibition of which leads to nuclear accumulation of PABPs after UV, which may in turn augment translational inhibition. ii Declaration I declare that the work presented in this PhD is my own and has not been submitted for any other degree. Hannah Burgess iii Acknowledgements Thanks to all members of the Gray lab past and present (except the frogs) for help and advice over the years. Special thanks to Matt for helping me become the lab’s best western blotter and being an excellent drinking buddy, and to Niki for everything, but especially for helping me get this thing written. Thanks to everyone at the HRSU who made us feel welcome. Many thanks to Shelia, Arantza and Mike for all the confocal and histology help, and for telling me my cells look pretty. Thanks to Marion, Rachel, G Lover and all the HGU students who made all the trips to Peebles, TGI Fridays, pirate parties and Christmas parties booze-fuelled mountains of fun… and a special mention to the Translation UK/CSH cool kids drinking team who may never be able stomach Tanqueray gin again. Thanks too to my far away friends - Cat, Phillipa, Natalie, Liz - whose visits were always much appreciated and big thanks to Sally for listening to my moans at the end of a day, letting me co-parent problem child Pingu and generally being an awesome flatmate and best mate. Finally, special thanks to Alistair for being the best reason to escape the lab on weekends and Mum and Dad for always telling me I could do it. iv Abbreviations 4EHP - 4E-homologous protein ARE – AU-rich element ARC - autoregulatory complex ARS - autoregulatory sequence ATP – adenosine triphosphate AGO - Argonaute protein BARD1 - BRCA1-associated RING domain protein 1 bp – base pairs BC1 – brain cytoplasmic 1 BRCA1 - Breast Cancer associated-1 CaMKIIα - calcium/calmodulin dependent protein kinase II α CBC - cap binding complex CBP – cap binding protein C/EBPβ - CCAAT/Enhancer β CHOP -C/EBP homologous protein CPEB - cytoplasmic polyadenylation element binding protein CPSF –cleavage and polyadenylation specificity factor CRM1 - chromosome region maintenance 1 CstF - cleavage stimulatory factor CTD - COOH-terminal domain of RNAPII DAB - 3,3’-diaminobenzidinetrahydrochloride DAPI - 4',6-diamidino-2-phenylindole DMEM - Dulbecco’s minimal essential medium DNA – deoxyribonucleic acid DRB – 5,6-dichlororibofuranosylbenzimidazole DSE - downstream sequence element DSP – Dithiobis (succinimidyl propionate) v EDTA – ethylene diamine tetraacetic acid eEF – eukaryotic elongation factor eIF – eukaryotic initiation factor eRF – eukaryotic release factor EJC – exon junction complex ELAV - embryonic lethal abnormal vision EMSA - electrophoretic mobility shift assay ER - endoplasmic reticulum FAK - focal adhesion kinase FISH – fluorescent in situ hybridisation FRAP - fluorescence recovery after photobleaching g – Grams g – centrifugal force G3BP - GTP-ase activating protein SH3 domain-binding protein GADD34 - growth arrest and DNA damage gene 34 GAP - GTPase activating protein GAPDH - glyceraldehyde-3-phosphate dehydrogenase GCN2 - general control non-depressible 2 GEF - guanine nucleotide exchange factor GFP – green fluorescent protein GGR - global genomic repair HA-tag – haemagglutinin HGNC - Hugo Gene Nomenclature Committee hnRNP - heterogeneous ribonucleoprotein HRI - haem-regulated inhibitor HRP – horseradish peroxidase HSV1 - herpes simplex virus type 1 IF - immunofluorescence vi IHC –immunohistochemistry IP - immunoprecipitation IPG – immobilised PH gradient IPTG - isopropyl β-D-1-thiogalactopyranoside IMP1 - insulin-like growth factor II mRNA binding protein-1 ITAF - IRES trans-acting factor Kb – kilobase kDa - kilodalton KSHV - Kaposi sarcoma-associated herpes virus KSRP - KH splicing regulatory protein L - litre LMB – leptomycin B LB – luria broth m7G - 7-methylguanosine M – molar MEF – mouse embryonic fibroblast ml – millilitre mM – millimolar miRISC - microRNA-induced silencing complex miRNA - Micro RNA mRNA – messenger RNA mTOR - mammalian target of rapamycin NAB2 - nuclear polyadenylated RNA-binding protein 2 NES – nuclear export signal NGD – no go decay NMD - nonsense-mediated decay NSD - non-stop decay NSP3 - non-structural protein 3 vii OPMD - oculopharyngeal muscular dystrophy ORF - open reading frame PABP – poly(A)-binding protein PABPN1 - Nuclear poly(A)-binding protein PAGE - polyacrylamide gel electrophoresis PAM – PABP-interacting motif PAP – poly(A) polymerase PBS – Phosphate buffered saline PCR – polymerase chain reaction PARN - poly(A)-specific ribonuclease PBS – phosphate buffered saline Pdcd4 - programmed cell death protein 4 PERK - PKR-like ER localized kinase PFA - paraformaldehyde pI – isoelectric point PI3K - phosphoinositde 3-kinase PKC - protein kinase C PKR - protein kinase activated by double-stranded RNA PP1 - protein phosphatase 1 PTC - premature termination codon PVDF - polyvinylidene fluoride RACK1 - Receptor for activated kinase 1 RNA – ribonucleic acid RNAi – RNA interference RNAPII – RNA polymerase II RNase - ribonuclease RNase MRP - RNase mitochondrial RNA processing RNP – ribonucleoprotein RoXaN - rotavirus X protein associated with NSP3 RRL - rabbit reticulocyte lysate viii RRM - RNA-recognition motif SAP - shrimp alkaline phosphatase SDS - sodium dodecyl sulphate SEM - standard error of the mean shRNA – short hairpin RNA SG – stress granule siRNA – short interfering RNA snoRNA - small nucleolar RNA TAP - Tip-association protein TBS - Tris-buffered saline TCA - trichloroacetic acid TCR - transcription coupled repair TD-NEM - transcription dependent nuclear export motif TEMED - tetramethylethylenediamine TGF-β - transforming growth factor beta TNF - tumour necrosis factor TREX - transcription/export tRNA - transfer RNA TTP – tristetraprolin UNR - upstream of N-ras UTR - untranslated region UV - ultraviolet VEGF - vascular endothelial growth factor VHL - von Hippel-Lindau VSV - vesicular stomatitis virus WB - western blot w/v – weight/volume YB-1 - Y-box binding protein 1 ix Table of contents Abstract........................................................................................................................i Declaration.................................................................................................................iii Acknowledgements....................................................................................................iv Abbreviations..............................................................................................................v Table of contents.........................................................................................................x List
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