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Thesis Corrections Investigations of the DEAD-box helicase eIF4A Nicola Marie Phillips, BSc., MSc. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy July 2011 Abstract Eukaryotic Initiation Factor (eIF) 4A is the most abundant initiation factor and the prototypical member of the DEAD-box family of helicases. Once recruited to the cap-binding complex, eIF4F, eIF4A unwinds inhibitory RNA secondary structure in the 5’ untranslated region (UTR) of mRNAs, promoting efficient ribosomal scanning to the start codon. The requirement for eIF4A in translation initiation correlates with increasing 5’ UTR length, suggesting that regulating the activity of eIF4A may affect the translation of particular mRNAs. It is well established that the transcripts of genes involved in cell cycle control and proliferation have long 5’ UTRs; therefore altering the activity of eIF4A may affect these genes specifically. A cross-discipline approach was used to investigate eIF4A helicase activity to obtain information regarding both the mechanics of helicase activity and the biological impacts of its inhibition. Recombinant eIF4A helicase activity, the stimulatory effect of eIF4B and the effect of known eIF4A inhibitors was first analysed using an ensemble helicase assay. Due to the limitations of this method a single molecule technique utilising optical tweezers was developed to investigate helicase activity at a higher force resolution. Optical tweezers were used to ‘trap’ and manipulate a dual-labeled RNA:DNA construct containing a central stem-loop hairpin known to be inhibitory to ribosomal scanning attached to functionalised microspheres. Although instrumental failure prevented the completion of these experiments, initial force extension curves using this molecule were obtained. Once established, this single molecule system may be used to observe eIF4A activity with its accessory protein eIF4B and known eIF4A inhibitors. 15-deoxy-delta(12, 14)-prostaglandin J2 (15d-PGJ2) is a newly identified natural inhibitor of eIF4A activity which induces apoptosis and is implicated in the resolution of inflammation. The translation of specific mRNAs affected by 15d- PGJ2 treatment of HeLa cells was analysed by translational profiling coupled to microarray analysis. No correlation, however, was seen between those transcripts that were translationally repressed and their 5’ UTR length or composition. i Acknowledgements Firstly I would like to thank my supervisors Keith Spriggs and Stephanie Allen for all their help and advice throughout my project. I would also like to thank Anne Willis, Martin Bushell, Lia De Moor and Catherine Jopling for access to their superior knowledge of science. Thanks to the University of Sussex, Korner travelling fund for enabling me to travel to Berlin to collaborate with JPK and to Joost Van Mameren and Anna Wozniak of JPK for their help using their optical tweezer systems and Phil Williams for helping me with the analysis of the data. Thanks to Jonas Emsley and Paul McEwan for the cells and vectors required for the Drosophila expression system and advice. Especial thanks to Keith who I feel has gone above and beyond the call of duty as a supervisor - I’ve loved working with you and 4A and I’m going to miss you both a lot. I would also like to thank him and his partner Hilary Collins for their help in the final, quite manic stage of handing this in. Thanks to everyone in the RNA biology group and LBSA past and present who have helped along the way, particularly Andrew Bottley for enthralling chats, Sasha Kondrashov for his endless knowledge and enthusiasm, Tiffany Hamilton and Kirsti Hill for advice and tissues, Helois Radford and Helen Booden for always being available for a pint, Laura Cobbold, Yi Wen Kong, Melissa Chen, Lucy Young and Helen King for providing ears aplenty and Andrew Lewis for always being available for duck walks. Thanks to my Mum and Dad who have always encouraged me and have been a huge source of support throughout the last few years (and all of my life come to think of it!). Thanks to my ‘inlaws’ Pam and Ed Blenkinsopp for providing lots of encouragement in addition to vast quantities of wine, chocolates and flowers throughout the years. Thanks to Sophie Cunningham and Michelle Ramsay for only being a phonecall away. Lastly, thanks to Aidan Blenkinsopp who has been a hero all along the way, I probably would not have started this without your encouragement and I definitely would not have finished it! You have been absolutely brilliant and I can’t thank you enough. ii Table of Contents Abstract ................................................................................................................................i Table of Contents............................................................................................................iii List of Figures ................................................................................................................. vii List of Abbreviations ..................................................................................................... ix 1 Introduction ................................................................................................................1 1.1 Helicases........................................................................................................................... 1 1.2 DEAD-box helicases ...................................................................................................... 2 1.2.1 Structure and conserved motifs.......................................................................................3 1.2.2 Mechanism ................................................................................................................................8 1.2.3 N and C terminal extensions........................................................................................... 11 1.3 eIF4A - the prototypical DEAD-box helicase is involved in translation ....12 1.4 Translation ....................................................................................................................13 1.5 Signaling pathways to translation .........................................................................15 1.6 The mechanism of eukaryotic translation ..........................................................16 1.7 The ribosome in translation ....................................................................................17 1.8 The mechanism of translation initiation.............................................................20 1.9 Cap-dependent translation initiation...................................................................22 1.10 Ribosomal subunit dissociation...........................................................................24 1.10.1 Formation of the 43S pre-initiation complex ....................................................... 25 1.10.2 Cap-recognition by eIF4F.............................................................................................. 26 1.10.3 Mechanism of 48S scanning to the start codon ................................................... 30 1.10.4 Identification of the start codon................................................................................. 32 1.11 Alternative mechanisms of translation initiation..........................................33 1.11.1 Alternative start codons ................................................................................................ 35 1.11.2 Ribosomal shunting......................................................................................................... 36 1.11.3 Internal Ribosomal Entry.............................................................................................. 37 1.11.4 5’ terminal oligopyrimidine tracts (TOPs)............................................................. 38 1.11.5 Regulation of translation initiation via 3’ UTR elements ................................ 38 1.12 eIF4A specific regulation........................................................................................40 1.13 eIF4G and homologues............................................................................................40 1.14 Other MIF4G/MA3 domain containing proteins/eIF4G competitors......44 1.14.1 Programmed cell death 4 .............................................................................................. 44 1.14.2 Nucleolar MIF4G-containing protein (NOM1) ..................................................... 46 1.14.3 15-deoxy-delta(12,14)-prostaglandin J2 ................................................................ 46 1.15 eIF4B..............................................................................................................................49 1.16 eIF4H .............................................................................................................................49 1.17 Other eIF4A binding proteins ...............................................................................51 1.17.1 RBM4...................................................................................................................................... 51 1.17.2 HuD ......................................................................................................................................... 51 1.17.3 Drosophila Mad/Medea.................................................................................................. 52 1.17.4 Cyclin dependent kinase (CDK) 4 .............................................................................. 53 1.17.5
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