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5-Methylcytidine Has a Complex, Context-Dependent Role in RNA

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5-Methylcytidine Has a Complex, Context-Dependent Role in RNA 5-methylcytidine has a complex, context-dependent role in RNA Andrew Mark Shafik April 2017 A thesis submitted for the degree of Doctor of Philosophy The Australian National University Originality Statement I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at ANU or any other educational institution, except where due acknowledgement is made in this thesis. Any contribution made to the research by others, with whom I have worked at ANU or elsewhere, is explicitly acknowledged in this thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. Signed ………………………. Date …………………………. II Acknowledgements I am overwhelmed. I have finally completed this milestone, three and a half years of my life culminating in this body of work. The path hasn’t been easy but I have finally reached the end. Of course, this would not have been possible without the support of many people. To my supervisor, Thomas Preiss, thank you for reading the thesis and the stimulating discussions. To Maurits Evers, thank you so much for the collaboration, for the bioinformatics support concerning the metagene analyses. This gave another dimension to the thesis. Thanks for reading my thesis, for the many valuable discussion we had, and for the coffee breaks. To Ulrike Schumann, thank you for your support and guidance. I really appreciate it. Thanks for reading my thesis and your positive feedback. To Yalin Liao and Eloisa Pagler, thanks for making me feel welcome and helping me to find my way around the lab. Thank you for being the voice of reason when times were tough. To Tennille Sibbritt, thanks for the support early on, and showing me some techniques in the lab. To Brian Parker thanks for bioinformatics support concerning the AGO2-m5C association. To other members of the lab, Rina Soetanto, Chikako Ragan and Nikolay Shirokikh thanks for the lab get-togethers and coffee breaks. To my Dad, Mum and sister. Many thanks for your unfailing and undying support. Thank you for being a soundboard when things got tough. I couldn’t have done this without you all. III Abstract Ribonucleic acid (RNA) metabolism processes and function are affected by specific RNA sequence motifs, the ability of RNA to form secondary structure and assemble into ribonucleoprotein (RNP) complexes (Dandekar & Bengert, 2002). As these aspects are likely to be affected by nucleoside modifications, it is important to document the extent and function of post-transcriptional modifications present in these molecules. Thus, there is an increasing focus on exploring the incidence and biological relevance of post-transcriptional marks in RNA. This thesis is aimed at defining a role for the post-transcriptional modification, 5- methylcytidine (m5C) particularly in mRNAs. Bisulfite treatment, which allows for the nucleotide-specific identification of m5C, coupled with next generation sequencing allowed for the detection of m5C sites transcriptome-wide at single-nucleotide resolution. This revealed candidate m5C sites in many RNA biotypes including both non-coding and coding RNA in cervical cancer cells (HeLa). In mRNAs, m5C candidate sites were observed in both untranslated regions and in the coding sequence, suggesting multiple roles for m5C. Global analyses revealed features of the m5C modification in nuclear-encoded mRNAs. m5C sites were shown to be relatively enriched in the 5′ UTR, to be associated with high GC content, low minimum free energy structures, and weakly translated mRNAs. Furthermore, experimental and bioinformatic approaches suggested a role for m5C in regulating mRNA translation by recruiting specific m5C-binding proteins, which were identified through a RNA bait pulldown approach. m5C was also suggested to function in stabilising target mRNA. Bioinformatic and experimental approaches indicated m5C might physically block the destabilising effect of Argonaute 2/microRNA binding. In this way, m5C would protect the transcript from degradation. Also, further work identified the m5C modification in mitochondrial (mt) rRNA, where a function of m5C is discussed, in novel structural positions in mt-tRNAs, but not in mt- mRNAs. Altogether, this thesis suggests that m5C has a complex, context-dependent role in RNA. IV Abbreviations used in this thesis Ψ Pseudouridine 5-aza-IP 5-azacytidine-mediated RNA immunoprecipitation AdoMet S-adenosyl L-methionine (SAM) AGO2 Argonaute 2 ALKBH α-ketoglutarate-dependent dioxygenase alkB homolog bp Base pairs BR Broad range BSA Bovine serum albumin bsRNA-seq Bisulfite RNA sequencing cDNA Complementary DNA CDS Coding sequence CIMS-miCLIP Cross-linking-induced mutation sites miCLIP CITS-miCLIP Cross-linking-induced truncation miCLIP CLIP Crosslinking and immunoprecipitation Co-IP Co-immunoprecipitation dH2O RNase, DNase free water dsDNA Double stranded DNA DNA Deoxyribonucleic acid DNase Deoxyribonuclease DNMT DNA methyltransferase DTT Dithiothreitol ECL Enhanced chemiluminescence EDTA Ethylenediamine tetracetic acid EZR Ezrin f5C 5-formylcytosine FBS Fetal bovine serum FDR False discovery rate FTO Fat mass and obesity associated protein GO Gene ontology hr Hours HEK293T Human embryonic kidney 293 cells HeLa Human cervical cancer cell line V HITS-CLIP High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation hm5C 5-hydroxymethylcytosine HuR Human antigen R IGV Integrated Genomics Viewer IP immunoprecipitation KD knockdown kDa Kilodalton LSU Large subunit m5C 5-methylcytidine m1A N1-methyladenosine m6A N6-methyladenosine LC-MS/MS Liquid chromatography mass spectrometry lncRNA Long non-coding RNA M Molar MeRIP Methyl-RNA immunoprecipitation METTL3 Methyltransferase like 3 METTL14 Methyltransferase like 14 miCLIP Methylated individual-nucleotide-resolution crosslinking and immunoprecipitation min Minute miRNA MicroRNA ml Millilitre mM Millimolar mRNA Messenger RNA mt Mitochondrial MTase Methyltransferase MYC Myelocytomatosis Viral Oncogene Homolog NaOAc Sodium acetate ncRNA Non-coding RNA ng Nanograms NGS Next generation sequencing nM Nanomolar Nop Nucleolar protein NSUN NOP2/Sun domain family, member VI nt Nucleotide NTC Non-targeting control P body Processing body PAR-CLIP Photoactivatable ribonucleoside-enhanced CLIP PBS Phosphate buffered saline PBST Phosphate buffered saline + Tween20 PCR Polymerase chain reaction Poly(A) Polyadenylation pre-miRNA Precursor miRNA pri-miRNA Primary miRNA R-Luc Renilla luciferase RBP RNA binding protein RCMT RNA (cytosine-5)-methyltransferase RDX Radixin Ribo-Seq Ribosome profiling sequencing RIPA Radioimmunoprecipitation assay buffer RISC RNA-induced silencing complex RNP Ribonucleoprotein RNA Ribonucleic acid RNA-seq RNA sequencing RNAi RNA interference RNase Ribonuclease RPKM Reads per kilobase of transcript per million mapped reads rRNA Ribosomal RNA RT Reverse transcriptase RT-qPCR Real time quantitative PCR SD Standard deviation SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis sec Seconds siRNA Small interfering RNA SNP Single nucleotide polymorphism snRNA Small nuclear RNA snoRNA Small nucleolar NRA SSU Small subunit TET Ten-eleven translocase VII TRDMT1 tRNA aspartic acid methyltransferase 1 tRNA transfer RNA µl Microlitres UV Ultraviolet UTR Untranslated region V Volts vtRNA Vault RNA WTAP Wilms tumor 1 associated protein w/v Weight per volume YTDHF YTH domain family VIII List of Figures Figure 1.1: Schematic illustrating the cap and internal modifcations known in mRNAs. 3 Figure 1.2: Schematics illustrating methods to detect m6A (immunoprecipitation) and m5C (bsRNA-seq, 5-Aza-IP, miCLIP). ........................................................................... 8 Figure 1.3: Schematic illustrating ‘writers’, ‘readers’ and ‘erasers’ of the m5C modification. .................................................................................................................. 12 Figure 1.4: Function of m6A and m5C in ncRNA (taken from my review: Shafik et al., 2016). .............................................................................................................................. 19 Figure 1.5: Schematic outlining the various roles of m6A in mRNA translation (adapted from my review: Schumann et al., 2016). ..................................................................... 22 Figure 1.6: Role of m1A in mRNA translation. .............................................................. 24 Figure 1.7: Examples where m5C methylation has both a repressive and enhancing affect on translation in mRNAs ..................................................................................... 27 Figure 3.1: Human mitochondrial genome ..................................................................... 55 Figure 3.2: BLAST search of human mitochondrial genome revealing a number of highly similar sequences to nuclear encoded genes ...................................................... 56 Figure 3.3: Quality control measures to ensure purity of mitoplast isolation. ............... 58 Figure 3.4: Illumina bsRNA-seq coverage across the mitochondrial transcriptome…...60 Figure 3.5: Integrated Genomics Viewer (IGV) screenshot of the called 16S m5C sites in the Illumina bsRNA-seq experiment ........................................................................
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