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Molecular regulation of mRNA stability and translation by Francisca Rojas Ringeling, MD A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland August 2017 c Francisca Rojas Ringeling, MD All rights reserved Abstract The flow of information from DNA to RNA to protein is a tightly regulated process, which ultimately determines the functional properties that each cell will possess. Defects in any of the multiple regulatory mechanisms that ensure that proper amounts of each protein are produced within a particular cell, may lead to dysregulation of cellular processes and disease. This dissertation will deal with 2 different projects. The common theme between these projects is that we studied post-transcriptional regulation of gene expression at the level of messenger RNA (mRNA), and in both studies we took advantage of genome-wide sequencing techniques to develop insights and novel hypothesis within the realm of neurobiology. Briefly, the first project is a study of the role of Cyfip1, a neuropsychiatric disease risk gene, in regulating the translation of its mRNA partners. We show that dele- tion and over-expression of Cyfip1 in the mouse brain, leads to diametric changes in protein translation of NMDAR subunits and postsynaptic components, and has consequences in behavior within these mouse models. The second study is an ex- ploration of the role of m6A epitranscriptional modification in the regulation of cortical neurogenesis in-vivo, where we identified a critical and conserved role of m6A in the temporal control of mammalian cortical neurogenesis. Advisor: Hongjun Song, PhD Reader: Guoli Ming, MD, PhD ii Acknowledgments I could not have written this thesis without the help of many people. Most notably, I thank my advisor Hongjun Song for his guidance and helpful advice throughout my doctoral studies. Hongjun has been a supportive and flexible mentor, and he encouraged me to learn new things and go beyond my comfort zone. I would also like to acknowledge Kijun Yoon, who has worked closely with me for the past 4 years. Kijun taught me almost everything I know about bench work (which was very close to zero when I started), and he was always patient and kind with me. I also learned a lot from him on how to develop scientific hypothesis and think critically about experimental designs, not to mention many fun discussions about movies, music, and many other topics. All the members of the Song Ming Lab have contributed to this thesis in one way or another. I really enjoyed working in our sometimes hectic, but always fun environment. I would like to thank the faculty of the Human Genetics Pre-doctoral Training Program, specifically Dr. David Valle for awarding me this wonderful opportunity. Also our program administrator Sandy Muscelli for all the work she does for us students. iii I am also much obliged to my thesis committee members Paul Worley, Guoli Ming and Andrew McCallion for their thoughtful comments and fruitful discus- sions. I would also like to express gratitude to my friends and colleagues who con- tributed to this thesis in many ways: Jessica Cassin, Caroline Vissers, Leire Abalde- Atrista´ın, Stephanie Temme, Caroline Siebald, Kai Kammers, Ursula Smole, Juan Calderon´ Giadrosic, Nam Nguyen, Ji Young Park, Dennisse Cyrus-Jimenez, Xinyuan Wang, and many others. You made Baltimore my home, and I feel very lucky to be able to call you my friends. I am deeply grateful to my wonderful parents and sister, who have always been supportive about everything I decide to do. I couldn’t be here if it weren’t for your relentless support. Finally, I want to thank my husband Stefan. I can’t express in words how lucky I am to have you. Thank you for being my partner in every aspect of our amazingly fun life. iv Contents 1 Introduction 1 2 Cyfip1 4 2.1 Abstract . 4 2.2 Introduction . 6 2.3 Methods . 8 2.3.1 Experimental model and subject details . 8 2.3.2 Method details . 9 2.3.3 Behavior studies . 13 2.4 Results . 17 2.4.1 Deletion of Cyfip1 results in behavioral abnormalities re- lated with schizophrenia . 17 2.4.2 Increased Cyfip1 dosage leads to ASD-like behavioral ab- normalities . 19 iv 2.4.3 Cyfip1 interacts with mRNAs of synaptic and NMDA re- ceptor related proteins in mouse hippocampus and human cerebral cortex. 22 2.4.4 Cyfip1 directly regulates mRNA translation of NMDAR subunits and postsynaptic components . 26 2.4.5 Protein expression of NMDAR subunits and postsynaptic components is altered within synaptosomes depending on Cyfip1 dosages . 28 2.4.6 Bidirectional modulation of NMDAR signaling rescues be- havioral abnormalities in cKO and cOE mice . 31 2.5 Discussion . 34 2.5.1 Cyfip1 mRNA targets are related to synaptic function and neuropsychiatric diseases . 35 2.5.2 Diametric gene dosage effects of CNVs in neurodevelop- mental diseases . 35 3 m6A controls cortical neurogenesis 37 3.1 Abstract . 37 3.2 Introduction . 39 3.3 Methods . 41 3.3.1 Experimental model and subject details . 41 3.3.2 Method details . 43 3.4 Results . 56 3.4.1 Nervous system Mettl14 deletion extends cortical neuroge- nesis into postnatal stages . 56 3.4.2 Mettl14 deletion in neural progenitor cells leads to pro- tracted cell cycle progression. 59 3.4.3 Mettl3 regulates embryonic cortical neurogenesis . 61 v 3.4.4 m6A tags transcripts related to transcription factors, cell cycle, and neurogenesis, and promotes their decay . 64 3.4.5 Mettl14 deletion uncovers transcriptional pre-patterning for normal cortical neurogenesis . 66 3.4.6 METTL14 regulates cell cycle progression of human corti- cal NPCs . 71 3.4.7 m6A-seq of human forebrain brain organoids and fetal brain reveals conserved and unique m6A landscape features com- pared to mouse . 73 3.5 Discussion . 76 3.5.1 Transcriptional pre-patterning for cortical neurogenesis . 76 3.5.2 Heightened transcriptional coordination of mammalian cor- tical neurogenesis by m6A . 77 3.5.3 Conserved and unique features of human m6A landscape during cortical neurogenesis . 78 A Supplemental Figures: Cyfip1 80 B Supplemental Figures: m6A 86 Curriculum 114 vi List of Figures 2.1 Cyfip1 mouse models. 18 2.2 Cyfip1 cKO shows behavior abnormalities related to schizophrenia. 20 2.3 Cyfip1 cOE mice show ASD-like behavioral abnormalities. 23 2.4 Cyfip1 RIP-seq representative coverage plots. 24 2.5 Gene ontology and disease ontology analysis of Cyfip1 target mR- NAs. 25 2.6 Confirmation of CYFIP1 mRNA targets in mouse hippocampus and human cerebral cortex. 27 2.7 Cyfip1 regulates translation of NMDAR subunits and postsynaptic components. 29 2.8 Altered protein expression of NMDAR subunits and postsynaptic components in synapses with differential Cyfip1 dosages. 30 2.9 Memantine treatment in cKO rescued behavioral abnormalities. 32 2.10 D-cycloserin treatment in cOE rescued behavioral abnormalities. 33 3.1 Nervous system Mettl14 deletion results in residual radial glia cells and ongoing neurogenesis in the postnatal mouse cortex. 57 vii 3.2 Mettl14−/− RGCs and NPCs exhibit prolonged cell cycle progres- sion. 60 3.3 Mettl14 cKO leads to depletion of m6A. 62 3.4 Mettl3 regulates cell cycle progression of NPCs and maintenance of embryonic cortical RGCs. 63 3.5 m6A tags transcripts related to transcription factors, cell cycle, and neuronal differentiation in the embryonic mouse brain. 65 3.6 m6A promotes mRNA decay. 67 3.7 Post-transcriptional regulation of pre-patterning gene levels by m6A methylation in cortical neural stem cells . 68 3.8 Regulation of protein production of pre-patterning genes by m6A methylation in cortical neural stem cells. 70 3.9 METTL14 regulates cell cycle progression of human NPCs. 72 3.10 Conserved and unique features of m6A mRNA methylation in hu- man forebrain organoids, human fetal brain and embryonic mouse forebrain. 75 A.1 Normal locomotor activity, motor coordination, nociception re- sponse and repetitive behaviors of Cyfip1 cKO mice. 81 A.2 Normal locomotor activity, novel object recognition, behavioral despair and sensorimotor gating of Cyfip1 cOE mice, and impaired maternal care of cOE mice. 82 A.3 Cyfip1 RIP seq strategy. 83 A.4 Bioinformatic analysis of Cyfip1 RIP seq experiment . 84 A.5 mRNA levels of Cyfip1 targets are unchanged in cKO and cOE mice. 85 B.1 Nervous system Mettl14 deletion in mice results in postnatal lethality. 87 viii B.2 Nervous system Mettl14 deletion in mice results in deficits in timely production of cortical neuron subtypes. 88 B.3 Flow cytometry analysis reveals delayed cell cycle progression of Mettl14−/− NPCs. 89 B.4 Mettl3 is essential for m6A mRNA methylation and proper cell cycle progression of mouse NPCs . 90 B.5 m6A-seq analysis of mouse embryonic forebrain. 91 B.6 Expression of neuronal genes in RGCs of embryonic cortex in vivo. 92 B.7 Mettl14 regulates cell cycle progression of hNPCs. 93 B.8 Comparison of m6A mRNA landscapes among human forebrain organoids, fetal brain and mouse embryonic forebrain. 94 ix CHAPTER 1 Introduction The flow of information from DNA to RNA to protein is a tightly regulated pro- cess, which ultimately determines the functional properties that each cell will pos- sess. Thus, it makes sense for the cell to have multiple levels of regulation of this flow of information, such as transcriptional regulation, differential isoform us- age, regulation by epitranscriptional modifications, regulation of messenger RNA (mRNA) degradation and localization, and regulation at the level of protein trans- lation. When any of these regulatory mechanisms goes astray, it can lead to dys- regulation of cellular processes and disease. The past couple of decades have seen a substantial improvement in our abil- ity to study the regulatory mechanisms involved in gene expression, owing mainly to the rapid advances in sequencing technologies and computational approaches to genomic data analysis.
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