Novel Bioinformatics Approaches for Microrna Detection and Target Prediction
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Novel Bioinformatics Approaches for MicroRNA Detection and Target Prediction by Subramanian Shankar Ajay A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Bioinformatics) in The University of Michigan 2009 Doctoral committee: Professor Brian D. Athey, Co-Chair Research Investigator Inhan Lee, Co-Chair Professor Daniel M. Burns Jr. Assistant Professor Zhaohui Qin Professor David J. States, University of Texas Health Sciences Center at Houston To amma, appa and panni ii Acknowledgements I would like to show my immense gratitude to Prof. Brian Athey and Dr. Inhan Lee who took me under their wings when I first joined the Bioinformatics doctoral degree program. Their enthusiasm has been so infectious that I derived confidence from it every time I interacted with them. They have been a pillar of knowledge and support during the course of my graduate studies. Their ideas have helped shape this thesis to a large extent and I am grateful to them for the encouragement that they continued to provide along every step of the way. I would like to thank Prof. David States for his invaluable inputs and discussions. The influence his pedagogical role has had in the first few years cannot be emphasized enough. Many thanks are also due to my doctoral committee members, Dr. Zhaohui Qin and Prof. Daniel Burns, for all their help when I turned to them. I have had the pleasure of learning bioinformatics methods and concepts under their tutelage. I really appreciate the fresh perspectives that Dr. Jeffrey DeWet lent when I had a tough time sorting out scientific problems, especially with respect to experiments. The extremely resourceful Bioinformatics graduate program staff, Alicia, Yuri, Julia, Sandy and Janet have helped make my transition and stay in the program facile. Much of the work in this thesis would not have been possible without the support from laboratories of Dr. Arul Chinnaiyan from the Department of Pathology at the iii University of Michigan, Dr. Haiming Chen from the Department of Psychiatry at the University of Michigan and Dr. JongIn Yook at Yonsei University. I am thankful to members of Dr. Chinnaiyan’s group, Mohan, Bharathy and Anju who have taught me experimental techniques, from using a pipette to cell transfections. Outside the scientific community some people have had an immeasurable impact in my life. The unconditional love and support (and money) from my family has seen me through highs and lows. The friends I’ve made through college and graduate school are ones I’ll forever stand by - the spasmers from BITS to the ommalites in Ann Arbor, among whom Jaggi and Sweta deserve a special mention for everything they’ve helped me with. Work would not have been as much fun without k2 and uthra, and lunches would not have been as entertaining without the discussions on everything from politics to movies with my colleagues at Green Court. I thank them all. iv Table of Contents Dedication........................................................................................................................... ii Acknowledgements............................................................................................................iii List of Figures..................................................................................................................viii List of Tables ..................................................................................................................... ix List of Appendices ..............................................................................................................x Chapter 1............................................................................................................................. 1 Introduction......................................................................................................................... 1 1.1 Post-transcriptional gene regulation ...................................................................... 2 1.2 Introduction to microRNAs ................................................................................... 3 1.2.1 MicroRNA biogenesis................................................................................. 4 1.2.2 Mechanisms of miRNA-mediated repression ............................................. 6 1.3 Nearest-neighbour thermodynamics...................................................................... 7 1.4 Problem Statement............................................................................................... 10 1.4.1 Detection of miRNA expression ............................................................... 11 1.4.2 Computational miRNA target prediction in animals................................. 13 1.5 Contributions ....................................................................................................... 15 1.5.1 Target-specific microarray probe design................................................... 15 1.5.2 Discovery of endogenous 5′-UTR target sites........................................... 16 1.6 Thesis Outline...................................................................................................... 18 Chapter 2........................................................................................................................... 19 Microarray probe design for miRNAs .............................................................................. 19 2.1 Background.......................................................................................................... 19 2.2 Computational Methods....................................................................................... 21 2.2.1 Base change strategy ................................................................................. 21 2.2.1 ProDeG algorithm ..................................................................................... 21 2.3 Computational Results......................................................................................... 24 2.3.1 Variance of Tm by introducing mismatches............................................... 24 2.3.2 ProDeG probes for human miRNA cDNAs .............................................. 25 2.3.3 Characteristics of ProDeG probes for cDNA of human miRNAs............. 26 2.3.4 ProDeG probes for RNA samples of human miRNAs.............................. 26 v 2.4 Experimental validation – Methods..................................................................... 27 2.4.1 Microarray platform .................................................................................. 27 2.4.2 let-7 family spiked-in experiments............................................................ 27 2.4.3 Hybridization experiment using lymphoblastoid cell-line small RNA ..... 28 2.4.5 Quantitative RT-PCR ................................................................................ 30 2.5 Experimental validation – Results....................................................................... 31 2.5.1 Verification of ProDeG cDNA probe specificity using let-7 spike-in experiments......................................................................................................... 31 2.5.2 Expression signals of ProDeG let-7 probes from human lymphoblastoid cell lines.............................................................................................................. 32 2.6 Discussion............................................................................................................ 33 Chapter 3........................................................................................................................... 47 Discovery of endogenous 5′-UTR miRNA target sites..................................................... 47 3.1 Background.......................................................................................................... 47 3.2 Results.................................................................................................................. 49 3.2.1 Presence of miRNA interaction sites in human 5′-UTR ........................... 49 3.2.2 hsa-miR-34a targets AXIN2 through both UTRs....................................... 50 3.2.3 Modified cel-lin-4 targets both lin28 UTRs .............................................. 52 3.3 Methods ............................................................................................................... 53 3.3.1 Bioinformatics and statistical analysis ...................................................... 53 3.3.2 Experimental validation – AXIN2 and hsa-miR-34a................................. 54 3.3.3 Experimental validation – LIN28 and lin-4 siRNA .................................. 55 3.4 Discussion............................................................................................................ 57 Chapter 4........................................................................................................................... 65 Post-transcriptional regulation by miRNA binding of uAUGs......................................... 65 4.1 Background.......................................................................................................... 65 4.2 Methods ............................................................................................................... 67 4.2.1 uAUG and miRNA sequence data............................................................. 67 4.2.2 Sequence complementarity search ............................................................ 68 4.2.3 miRNA expression data............................................................................