The role of polyadenylation in the induction of inflammatory genes Raj Gandhi BSc & ARCS Thesis submitted for the degree of Doctor of Philosophy September 2016 Declaration Except where acknowledged in the text, I declare that this thesis is my own work and is based on research that was undertaken by me in the School of Pharmacy, Faculty of Science, The University of Nottingham. i Acknowledgements First and foremost, I give thanks to my primary supervisor Dr. Cornelia de Moor. She supported me at every step, always made time for me whenever I needed it, and was sympathetic during times of difficulty. I feel very, very fortunate to have been her student. I would also like to thank Dr. Catherine Jopling for her advice and Dr. Graeme Thorn for being so patient and giving me so much help in understanding the bioinformatics parts of my project. I am grateful to Dr. Anna Piccinini and Dr. Sadaf Ashraf for filling in huge gaps in my knowledge about inflammation and osteoarthritis, and to Dr. Sunir Malla for help with the TAIL-seq work. I thank Dr. Richa Singhania and Kathryn Williams for proofreading. Dr. Hannah Parker was my “big sister” in the lab from my first day, and I am very grateful for all her help and for her friendship. My project was made all the more enjoyable/bearable by the members of the Gene Regulation and RNA Biology group, especially Jialiang Lin, Kathryn Williams, Dr. Richa Singhania, Aimée Parsons, Dan Smalley, and Hibah Al-Masmoum. Barbara Rampersad was a wonderful technician. Mike Thomas, James Williamson, Will Hawley, Tom Upton, and Jamie Ware were some of the best of friends I could have hoped to make in Nottingham. Regularly meeting up for board games nights was instrumental in the maintenance of my sanity. I am thankful to Jess Beaver, my personal trainer and friend, through whom I discovered my hobby of fitness and formed goals that gave me a sense of purpose and meaning. Yes, my life is that sad. I give thanks to my girlfriend Janay Gibbons for helping me to keep my head screwed on towards the end when it started loosening. Lastly, I am grateful to my parents and I thank my beloved siblings Vikram Gandhi, Krishna Gandhi, and Catherine Soskice-Gandhi for their love and support, especially through tough times. i Abstract Polyadenylation is a universal step in the production of all metazoan mRNAs except histone mRNA. Despite being universal, previous experiments have implicated it in the regulation of inflammation. An inflammatory system using RAW 264.7 murine macrophage cells was established with bacterial lipopolysaccharide (LPS) used as a stimulus. After improving the poly(A) tail test (PAT) method of measuring poly(A) tail lengths, it was applied to inflammatory mRNAs during the inflammatory response. Poly(A) tail length was shown to vary over the course of the inflammatory response, and for Tnf, this was even true of initial poly(A) tail size, which is widely believed to be uniform for the majority of mRNAs. The adenosine analogue cordycepin (3’- deoxyadenosine) was shown to have anti-inflammatory effects on mRNA, in line with existing literature, and is likely to be the anti-inflammatory component of Cordyceps militaris ethanol extract. Inhibition of either import of cordycepin into cells or phosphorylation of cordycepin was sufficient to abolish its anti-inflammatory effects. Adenosine treatment led to repression of Il1b mRNA, but did not repress other mRNAs tested that were cordycepin-sensitive. This suggests that cordycepin does not simply act by mimicking the effect of adenosine, and that the two compounds have distinct modes of action. Inhibiting deamination of cordycepin potentiated its effects. We also observed that pre-mRNA levels of inflammatory genes were decreased by cordycepin treatment, indicative of effects on transcription. Other groups have reported that cordycepin interferes with NF-B signalling. As NF-B is an important transcription factor for the induction of inflammatory genes, this would provide a basis for explaining our observation that cordycepin represses at the transcriptional level. However, we did not observe any changes in NF-B signalling, with degradation of IB completely unimpeded by cordycepin treatment. Notably, cordycepin did shorten the Tnf poly(A) tail, and the ii observed inhibition of polyadenylation is consistent with observations that cordycepin led to decreased efficiencies of mRNA 3’ cleavage and transcription termination for Tnf. Such effects on polyadenylation and 3’ processing of mRNA were hypothesised to particularly affect unstable mRNAs that depend on longer poly(A) tails for avoiding decay and/or mRNAs with a high rate of transcription. However, comparison of microarray data to data from RNA-seq of RNA from 4- thiouridine labelling experiments showed that cordycepin-sensitivity did not correlate with mRNA stability or transcription rate. Long noncoding RNAs (lncRNAs) were found to be enriched in cordycepin-treated cells. If some of those lncRNAs have regulatory roles in inflammation, cordycepin’s effects may be mediated through them. Lastly, cordycepin significantly altered pain behaviour in a rat model of osteoarthritis (OA), supporting its continued use as a lead compound for exploration of new OA therapeutics. iii Abstract .......................................................................................................................... i List of figures ............................................................................................................... vii List of tables ................................................................................................................. xi List of abbreviations .................................................................................................... xii 1 Introduction .......................................................................................................... 1 1.1 Polyadenylation ............................................................................................ 2 1.1.1 Mechanics of nuclear polyadenylation ................................................. 2 1.1.2 Control of poly(A) tail length ................................................................ 5 1.1.3 Poly(A) polymerases and other types of polyadenylation .................... 8 1.1.3.1 Cytoplasmic polyadenylation .......................................................... 10 1.1.3.2 Mitochondrial polyadenylation....................................................... 12 1.1.4 Biological importance and role of the poly(A) tail .............................. 13 1.1.5 Alternative polyadenylation................................................................ 17 1.1.6 Measuring poly(A) tails ....................................................................... 17 1.2 Cordycepin .................................................................................................. 24 1.2.1 Cordycepin and transcription ............................................................. 24 1.2.2 Cordycepin and mTOR signalling ........................................................ 25 1.2.3 Cordycepin and polyadenylation ........................................................ 27 1.2.3.1 Polyadenylation and cordycepin’s mechanism ............................... 29 1.3 Inflammation............................................................................................... 30 1.3.1 Inflammation and pain ........................................................................ 31 1.3.2 Macrophages ...................................................................................... 32 1.3.2.1 Macrophage receptors and signalling ............................................. 34 1.3.2.1.1 LPS signalling through TLR4 ...................................................... 35 1.3.2.1.1.1 MyD88-dependent pathway .............................................. 36 1.3.2.1.1.2 TRIF-dependent pathway .................................................. 38 1.3.2.1.2 NF-κB signalling in inflammation .............................................. 39 1.3.2.1.3 Regulation of inflammatory cytokine mRNA stability .............. 42 1.3.3 Anti-inflammatory effects of cordycepin ............................................ 45 1.4 Osteoarthritis – a therapeutic application of cordycepin? ......................... 47 1.5 Project aims and outcomes ........................................................................ 51 2 Materials and methods ....................................................................................... 53 iv 2.1 Cell work ..................................................................................................... 53 2.1.1 Cell culture .......................................................................................... 53 2.1.2 Cell stimulation with lipopolysaccharide (LPS) and treatments with compounds ......................................................................................................... 53 2.1.3 Preparation of fungal ethanol extracts for use in cell culture ............ 54 2.1.3.1 Assessing cordycepin and 3’ deoxyinosine concentration in fungal extracts by liquid chromatography/mass spectrometry (LC/MS) (performed by Wahyu Utami) ............................................................................................ 55 2.2 RNA work .................................................................................................... 56 2.2.1 RNA isolation ....................................................................................... 56 2.2.2 RNA isolation