STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF NONCODING RNAS IN MAMMALIAN CELLS by Sungyul Lee A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland December, 2015 © 2015 Sungyul Lee All Rights Reserved Abstract Francis Crick proposed the central dogma of molecular biology more than a half century ago focusing on the role of RNA as a messenger which delivers genetic information from DNA to protein. However, it is now clear that RNA constitute a major player in every aspects of biological processes as much as protein does, through their noncoding functions. While early studies of RNA biology were mostly centered around abundant and constitutive noncoding RNAs in ribosome, spliceosome, transcriptional machinery and telomere, recent studies are now shifting their heads toward less abundant and dynamically regulated tissue or developmental time specific noncoding RNAs such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). With advent of new analytic tools and massive amount of sequencing data, there have been continued unexpected discoveries revealing how our genome is written and read inside the cell. MicroRNAs are ~22 nt small RNAs that guide RISC proteins to their target genes through their base complementary thereby achieve posttranscriptional gene repression. The mechanism of repression is almost universal in animals but the regulation of their expression is one of big questions in the field. In order to facilitate investigations of expression control of miRNAs in mammals, we annotated genome-wide primary miRNA transcripts of mouse and human. We undertook this endeavor to provide most comprehensive transcriptional pictures across human and mouse genomes, which is a major bottleneck in the elucidation of mechanisms that controls miRNA abundance. To do this, we had to overcome 3 obstacles. First, we expressed dominant-negative DROSHA mutant to suppress efficient hairpin cropping of microprocessor thefore enriched un-processed primary transcripts for sequencing. Second, we used panel of ii human and mouse cell lines of diverse origin to increase coverage of miRNAs that are expressed tissue specifically. Lastly, we collaborated with Steven Salzberg’s lab to employ recently developed assembly algorithm, StringTie, which outperforms other existing assembly tools for this application. Together these, we uncovered unanticipated features and new potential regulatory mechanisms, including link between pri-miRNAs and distant mRNAs, and alternative splicing and alternative promoter usage that can produce transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results provide a valuable resource for the study of mammalian miRNA regulation. Another class of emerging regulatory noncoding RNA is long noncoding RNA (lncRNA). Although current human genome annotation predicts almost similar number of genes encoding lncRNA as protein coding genes, the question remains how many of them are indeed plays integral part of diverse biological functions. Unlike miRNA, mechanisms of lncRNAs are quite unique in each case, making it difficult to predict their function based on primary sequence. One of very limited number of ways to find their functions is to investigate their phenotype in cellular or organismal level after introducing genetic ablation. Through the screening of lncRNA that are induced after DNA damage, we identified NORAD which suggested its functionality given their high conservation in mammals, high abundance, and association with an interesting biological cue (i.e. induction after DNA damage). Surprisingly, cells inactivated NORAD expression showed increased level of numerical and structural chromosomal instability. We found this transcript harbors unusually high number of PUMILIO binding motifs allowing it to sequester this RNA binding protein (RBP), thereby suppressing its repressive activity on its targets. PUMILIO targets includes factors important for DNA damage response, DNA iii repair, and mitosis. Overexpression of PUMILIO also showed suppression of these target genes and phenocopied NORAD knockout cells. I also generated knockout mouse of clear NORAD ortholog Norad, using CRISPR/Cas9 technology. It might be very interesting to see the same phenotype in this animal, and possibly other phenotypes that we couldn’t observe due to simplicity of cultured cells. Altogether this study shows novel mechanism of genomic stability maintenance through sequestrating PUMILIO by a lncRNA, NORAD. Advisor: Joshua T. Mendell, M.D., Ph.D. Reader: Ben Ho Park, M.D., Ph.D. and Haig H. Kazazian, M.D., Ph.D. iv Preface My dissertation work written in this book only partially reflects what I was given and supported from wonderful people and institutions around me. Without helps and influences from them, this work could never been materialized. First and foremost, I’m immensely grateful to my mentor Joshua Mendell, and I’m truly indebted for his scientific acumen and critical thinking. His enthusiasm for unknowns and pursuit of perfection always inspired me and motivated my scientific creativity. I believe his influence and legacy will continue to be remained on my future career. My thesis committee members, Haig Kazazian and Ben Ho Park provided me valuable guidance throughout my thesis work. I could only continue to be professionally nurtured through our annual meetings, with their constructive criticisms and solutions for problems each time I had. I also thank my colleagues in Mendell lab. In particular, Tsung-Cheng Chang taught me so many useful experimental technics and Florian Kopp was always there with me to discuss and perform exciting works together. I thank my graduate program Pathobiology at Johns Hopkins for giving me administrative and financial support. Lab manager Ana Doughty was the most helpful people I ever met and Molecular biology department in UT Southwestern enabled me to continue my work in Dallas. I thank our excellent collaboration groups including Stephen Salzberg lab, Yang Xie lab, and Hongtao Yu lab. Finally, I can’t finish my acknowledgements without saying thank you to my family. My parents Se-il and Young-sook inherited in me their appreciation of hard-work and thankfulness for everything happening around me. My proud son, Shihoo is the energy that always drives me go and the best motivation of my life. This dissertation is dedicated to Jung Hee, mother of my son and wife of mine, who shares every sorrows and joys of my life with me. v Table of Contents Abstract ........................................................................................................................... ii Preface ............................................................................................................................ v Table of Contents ........................................................................................................... vi List of Tables ................................................................................................................. vii List of Figures ............................................................................................................... viii Chapter 1: Introduction ................................................................................................... 1 Chapter 2: Genome-wide annotation of microRNA primary transcript structures ...........10 Introduction ................................................................................................................10 Results .......................................................................................................................14 Discussion .................................................................................................................61 Materials and methods ...............................................................................................64 Chapter 3: Characterization and loss of function study of a human long noncoding RNA induced by DNA damage, NORAD ................................................................................74 Introduction ................................................................................................................74 Results .......................................................................................................................77 Discussion ............................................................................................................... 107 Materials and Methods ............................................................................................. 109 Chapter 4: Mechanism of chromosome instability in NORAD depleted cells ................ 120 Introduction .............................................................................................................. 120 Results ..................................................................................................................... 123 Discussion ............................................................................................................... 156 Materials and Methods ............................................................................................. 161 Chapter 5: Generation of Norad knockout mouse using CRISPR/Cas9 genome editing system ......................................................................................................................... 175 Introduction .............................................................................................................