Bioinformatics approaches to analysing RNA mediated regulation of gene expression Dissertation zur Erlangung des akademischen Grades "doctor rerum naturalium" (Dr. rer. nat.) eingereicht im Institut für Biochemie und Biologie an der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Potsdam LIAM CHILDS Arbeitsgruppe Bioinformatik Max-Plank-Institut für Molekulare Pflanzenphysiologie Potsdam, den 13.07.2009 This work is licensed under a Creative Commons License: Attribution - Noncommercial - Share Alike 3.0 Germany To view a copy of this license visit http://creativecommons.org/licenses/by-nc-sa/3.0/de/deed.en Published online at the Institutional Repository of the University of Potsdam: URL http://opus.kobv.de/ubp/volltexte/2010/4128/ URN urn:nbn:de:kobv:517-opus-41284 http://nbn-resolving.org/urn:nbn:de:kobv:517-opus-41284 Acknowledgements Dirk Walther is certainly one of the best supervisors I’ve ever had. He gave me the freedom to pursue my own ideas and goals whilst providing all the necessary help and support when needed. I have a sneaking suspicion that, if I didn’t play the drums, I may not have been chosen to fill this PhD position. Nevertheless, studying under Dirk and playing in a kick-arse rock/pop/punk/funk/blues/jazz band together has made the past three years, although so very far from home, a very happy and fulfilling experience. To work at such an institute, in the company of great scientific minds, has helped my scientific thinking through a colossal improvement. I still have far to go, but I start my post-doctoral efforts from a very solid basis. I would also like to thank my official university supervisor Joachim Selbig. Thanks to the whole of AG Bioinformatics; Christian Schudoma, Zoran Nikoloski and Patrick May for all the help provided, and also to Manuela Hische, Sergio Grimbs, Sebastian Klie, Georg Basler, Jan-Ole Christian, Tanja Gärtner, Henning Redestig, Pawel Durek, Jan Hummel and Xiaoliang Sun for the good times and great company. Thanks also to Thomas Altmann and Hanna Witucka-Wall for their help and for my involvement in their project along with Karl Schmid and Torsten Günther. Quick thanks to Marco Ende and Peter Krüger for technical support. Thanks to two very special friends Jan Lisec and Sandra Witt for the company, both wonderfully immature and entertaining, at lunchtime and during the jamming sessions. I thank my family for their support; Mum, Dad and Owen, and Sean for shedding a little brilliance on my projects whenever I was stuck. 16,110 km is no small distance and I miss you all very much. And finally, to my girlfriend Julia, my thanks to you can not be written down on paper ;). iii Selbständigkeitserklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig und unter Verwendung keiner anderen als den von mir angegebenen Quellen und Hilfsmitteln verfasst habe. Ferner erkläre ich, dass ich bisher weder an der Universität Potsdam noch anderweitig versucht habe, eine Dissertation einzureichen oder mich einer Doktorprüfung zu unterziehen. Potsdam, 13 July 2009 ____________________________________ Liam Childs v Contents Abstract ................................................................................................................................................... xi Lay Abstract .................................................................................................................................................. xii Zusammenfassung .............................................................................................................................................. xiv Chapter 1. Introduction ........................................................................................................................................ 1 1.1. What is RNA? .............................................................................................................................................. 2 1.1. The Central Dogma .................................................................................................................................... 2 1.2. The RNA world hypothesis .......................................................................................................................... 3 1.3. Discovery of new ncRNA ............................................................................................................................ 4 1.3.1. miRNA .................................................................................................................................................. 5 1.3.2. snoRNA ................................................................................................................................................ 6 1.3.3. Riboswitches......................................................................................................................................... 7 1.3.4. Ribozymes ............................................................................................................................................ 8 1.3.5. Artificial ncRNA ................................................................................................................................... 9 1.4. Arabidopsis ................................................................................................................................................. 9 1.4.1. ncRNA in Arabidopsis .......................................................................................................................... 9 1.5. Key concepts ............................................................................................................................................. 11 1.5.1. Folding RNA ...................................................................................................................................... 11 1.5.2. Graphs and graph theory ................................................................................................................... 14 1.5.3. State machines.................................................................................................................................... 15 1.5.4. Hidden Markov Models ...................................................................................................................... 15 1.5.5. Covariance models ............................................................................................................................. 16 1.5.6. Microarrays ....................................................................................................................................... 17 1.5.7. Support vector machines .................................................................................................................... 17 1.5.8. Association studies ............................................................................................................................. 18 1.6. Outline ...................................................................................................................................................... 19 Chapter 2. Arabidopsis genotyping .................................................................................................................... 21 2.1. Abstract ..................................................................................................................................................... 21 vii 2.2. Introduction .............................................................................................................................................. 22 2.3. Methods ..................................................................................................................................................... 25 2.3.1. Selection of accessions ....................................................................................................................... 25 2.3.2. Plant material preparation for phenotypic analysis .......................................................................... 25 2.3.3. Plant material preparation for genotypic analysis ............................................................................ 27 2.3.4. SFP identification .............................................................................................................................. 28 2.3.5. Analysis of functional gene categories ............................................................................................... 30 2.3.6. Detection of duplications and deletions ............................................................................................. 30 2.3.7. Whole genome haplotyping ................................................................................................................ 31 2.3.8. Identification of selective sweeps ....................................................................................................... 31 2.3.9. SFP-trait associations ........................................................................................................................ 32 2.4. Results ....................................................................................................................................................... 33 2.4.1. Patterns of SFP diversity ................................................................................................................... 33 2.4.2. Deletions and duplications ................................................................................................................. 37 2.4.3. Population structure analysis ...........................................................................................................