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Revising the Evolutionary Imprint of Rna Structure in Mammalian Genomes

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Revising the Evolutionary Imprint of Rna Structure in Mammalian Genomes REVISING THE EVOLUTIONARY IMPRINT OF RNA STRUCTURE IN MAMMALIAN GENOMES MARTIN ALEXANDER SMITH B.SC, M.SC A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2012 Institute for Molecular Bioscience ABSTRACT HE COMPLETE HUMAN GENOME SEQUENCE has been resolved for more than T a decade, yet the fraction that is biologically functional remains uncertain. Classical genes encoding proteins compose less than 2% of the sequence, while the majority is transcribed in dynamic and specific patterns during differentiation and development. Hence, the pervasive nature of mammalian transcription lies in stark contrast to its functional annotation. The key to resolving this discrepancy likely resides in the structural and functional analysis of the extensive non-protein cod- ing regions of the genome, which scale proportionately to organismal complexity throughout metazoan evolution. Moreover there is increasing, albeit still limited, evidence that non-protein-coding RNA transcripts arising from the genome convey a multitude of functional roles in the cell, primarily in the regulation of epigenetic processes. This thesis investigates the structures of RNA molecules encoded in mam- malian genomes, with the aim of characterizing the biological foundation of the expansive genomic landscape of undetermined function. In the first part of the thesis I describe the structural predictions of non-protein coding RNAs involved in the regulation of mammary development, breast cancer, melanoma, and mitochon- drial disease. These findings support the idea that non-protein coding portions of mammalian genomes may indeed convey function through RNA structure, and are presumably therefore subject to evolutionary selection. However, these non-coding regions only display patchy evidence of sequence conservation, consistent with observations that less than v10% of the mammalian genome sequence is conserved throughout evolution. These studies do not consider RNA structure conservation, which adheres to distinct evolutionary dynamics than when the function of a given locus is derived from sequence constraint alone. Indeed, genomic loci that function through RNA structure display greater evolutionary plasticity than, for instance, protein-coding regions as mutations may be incorporated more liberally, so long as they maintain base-pairing in the entailing structure. Past reports premised on evaluating RNA structure conservation are limited in the breadth of sampled loci and produce an unsettling amount of false positives and divergent predictions. ii I address these methodological impediments in the subsequent portions of this thesis through the development of an algorithm for benchmarking the performance of RNA secondary structure prediction. Two refined, energy-based consensus structure prediction algorithms (RNAZ and SISSIZ) were tested on a broad set of true positives that reflects experimental methodologies, which enables indepen- dent performance evaluation under variable parameters. The results expose the complementary nature of both algorithms, highlighting SISSIZ’s strength at detect- ing evolutionary conserved RNA structures where sequence conservation is limited. I subsequently describe the elaboration of a hybrid algorithm for massively parallel, comparative genomic screens of RNA secondary structure conservation based on the optimal performance range of each tested algorithm, as determined from the aforementioned benchmarking. When applied to consistency-based multi- ple genome alignments of 35 mammals, this approach confidently identifies over 4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails a false discovery rate of 8.1%, a historic low for such analyses. These predictions comprise 13.6% of the human genome, 88% of which fall out- side any known sequence constrained element. The findings divulged in this thesis represent a lower bound; extrapolations suggest that over 30% of the genome is un- der natural purifying selection for RNA structure, consistent with other indices that suggest that a large proportion of the mammalian genome is functional. The ex- tensive set of functional transcriptomic annotations presented in this thesis provide a comprehensive resource to aid in uncovering the precise molecular mechanisms underlying complex diseases, development and evolution, as well as the beginnings of a potential basis for parsing structure-function relationships in the vast numbers of noncoding RNAs expressed from mammalian genomes. iii DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copy- right permission from the copyright holder to reproduce material in this thesis. iv PUBLICATIONS DURING CANDIDATURE Peer-reviewed journal articles: • Khaitan D, Dinger ME, Mazar J, Crawford J, Smith MA, Mattick JS, and Per- era RJ. The melanoma-upregulated long noncoding RNA SPRY4-IT1 modu- lates apoptosis and invasion. Cancer Research 71:3852 2011 • Askarian-Amiri ME, Crawford J, French JD, Smart CE, Smith MA, Clark MB, Ru K, Mercer TR, Thompson ER, Lakhani SR, Vargas AC, Campbell IG, Brown MA, Dinger ME, and Mattick JS. SNORD-host RNA Zfas1 is a regulator of mammary development and a potential marker for breast cancer. RNA 17:878–891 2011 • Mercer TR, Neph S, Dinger ME, Crawford J, Smith MA, Shearwood AMJ, Haugen E, Bracken CP, Rackham O, Stamatoyannopoulos JA, and Mattick JS. The human mitochondrial transcriptome. Cell 146:645–658 2011 • Shore AN, Kabotyanski E, Roarty K, Smith MA, Dinger ME, and Rosen JM. Pregnancy-induced noncoding RNA (PINC) associates with PRC2 and regu- lates mammary epithelial differentiation. PLoS Genetics 8:e1002840 2012 Manuscripts currently under peer-review: • Smith MA, Gesell T, Stadler PF, and Mattick JS. Widespread purifying se- lection on RNA structure in mammals. Submitted to PLoS Genetics Web resources: • http://www.martinalexandersmith.com/ECS Software and data presented in Chapters 3 & 4. v PUBLICATIONS INCLUDED IN THIS THESIS Primary contributions: Smith et al. (2012) – incorporated as Chapters 3 and 4 Author Contribution Smith, M. A. (candidate) 100% Designed experiments 80% Wrote and edited manuscript 70% Analyzed results 50% Conceived study Gesell, T. 20% Analyzed results Revised underlying software Stadler, P. F. 20% Conceived study 10% Analyzed results 10% Wrote and edited manuscript Mattick, J. S. 30% Conceived study 10% Wrote and edited manuscript Secondary contributions: Publication Candidate’s contribution Khaitan et al. (2011) All 2D RNA structure analyses Incorporated into Chapter 2 Askarian-Amiri et al. (2011) All 2D RNA structure analyses Incorporated into Chapter 2 Shore et al. (2012) All 2D & 3D RNA structure analyses Incorporated into Chapter 2 Mercer et al. (2011) All 2D RNA structure analyses Incorporated into Chapter 2 vi CONTRIBUTIONS BY OTHERS TO THE THESIS No contributions by others. STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE None vii ACKNOWLEDGEMENTS HIS THESIS WOULD NOT HAVE BEEN POSSIBLE without the guidance and T support of my supervisors, Prof John S. Mattick and Prof Peter F. Stadler. Their valuable mentorship has provided me with enlightenment and confidence in addition to the refinement of my scientific skills. It is a pleasure for me to thank my thesis advisory group for their consistent feedback and constructive criticism, as well as the examiners of this thesis for their time and consideration. I would also like to thank my colleagues Dr Tim Mercer and Dr Marcel Dinger for their invaluable assistance, collaboration, and general camaraderie during my doctoral studies, in addition to all members and visitors of the Mattick group and the Leipzig centre for bioinformatics. I owe much respect to Dr Amanda Carozzi for her superhuman skills as a postgraduate administrative officer. I am also obliged to the University of Queens- land and the Australian and Queensland governments for providing the financial and infrastructural support that permitted me to undertake my PhD studies and to complete this thesis. My parents and their partners are also to thank for their constant support and understanding throughout my academic career. Very
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