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The Role of Dsrna in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena Thermophila Jason Motl Washington University in St

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The Role of Dsrna in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena Thermophila Jason Motl Washington University in St Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) 1-1-2011 The Role of dsRNA in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena thermophila Jason Motl Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Motl, Jason, "The Role of dsRNA in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena thermophila" (2011). All Theses and Dissertations (ETDs). 623. https://openscholarship.wustl.edu/etd/623 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY Division of Biology and Biomedical Sciences Molecular Genetics & Genomics Dissertation Examination Committee: Douglas L. Chalker, Chair Sarah C. R. Elgin Susana Gonzalo-Hervas John E. Majors Tim B Schedl Sheila A. Stewart THE ROLE OF dsRNA IN NUCLEAR DIFFERENTIATION AND REMODELING IN THE CILIATE, TETRAHYMENA THERMOPHILA by Jason Andrew Motl A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2011 Saint Louis, Missouri ABSTRACT OF THE DISSERTATION The Role of dsRNA in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena thermophila by Jason Andrew Motl Doctor of Philosophy in Biology and Biological Sciences (Molecular Genetics and Genomics) Washington University in St. Louis, 2011 Professor Douglas L. Chalker, Chairperson The ciliate, Tetrahymena thermophila, like a handful of other eukaryotes, engages in massive genome reorganization known collectively as chromatin diminution. Part of this process involves large-scale DNA excision known as DNA elimination. Recent data has shown DNA elimination to be dependent on RNA interference (RNAi). Using T. thermophila, I have sought to determine the role of non-coding RNA (ncRNA) in RNAi- dependent DNA elimination through studies of DNA sequences that are to be eliminated called internal eliminated sequences (IESs) and through a conjugation-specific Dicer protein and its putative tandem dsRNA-binding motif (DSRM) protein partners. Studies of the R IES revealed the requirement of IES DNA for production of long, bidirectional ncRNA early in conjugation. This ncRNA is essential for IES excision in zygotic nuclei later in conjugation. The conjugation-specific Dicer homologue, DCL1, was shown to be required for production of a species of sRNA called scnRNAs from the long, ii bidirectional ncRNA from IESs. Knockouts of DCL1 displayed a loss of these scnRNAs as well as an increase in the long, bidirectional ncRNA precursors. A deficiency in these scnRNAs was sufficient to block modification of chromatin associated with IESs and prevent their rearrangement later in conjugation. Failure of DNA elimination caused DCL1 knockout cells to arrest before completion of conjugation. Further studies of the tandem DSRM-containing proteins, DRB2 and DRB1, revealed that neither are solely partners for DCL1 or any other Dicer protein but play other important roles during conjugation. Zygotic expression of DRB2 was shown to be essential for DNA elimination and completion of conjugation. Interaction with the chromo-domain containing protein, Pdd1p, by Drb2p implicates ncRNA or sRNA in later stages of conjugation after scnRNA production. Knockouts of the tandem DSRM-containing DRB1 caused higher numbers of cells to abort conjugation and therefore produce fewer progeny. Localization of this protein to the crescent micronucleus during prophase of meiosis I links DRB1 to a probable role in ensuring proper recombination during meiosis for haploid gamete production. All these studies suggest that ncRNA has many roles in conjugation-specific processes including RNAi-directed DNA elimination. iii ACKNOWLEDGEMENTS I would like to thank all the current members of the Chalker lab, Annie Shieh, Scott Horrell, Christine Carle and Rachel Schwope, for their support when things went wrong, for listening and helping when I had questions and for producing a lab environment conducive to research (even when no experiments were working). In addition I would like to thank Scott Horrell for taking the time to proofread portions of this dissertation. I would also like to thank all the past members of the Chalker lab. In particular, I would like to thank Colin Malone and Alissa Anderson for all their help with the DCL1 knockout project and for making the Chalker lab feel like a home, in which I could do research. Next I would like to thank my thesis committee for their probing questions and excellent advice to further this research and produce the best data in the finite amount of time I had. In particular I would like to show my deep gratitude to my thesis advisor Doug Chalker for showing the patience and flexibility I needed to do research while trying to balance work with a growing young family. Given a different thesis advisor I do not think I could have completed these research projects and this dissertation. Thank you Doug. Lastly I would like to thank my wife, Jen, and my children, Kaya and Nathan, for supporting me through graduate school and lifting me up after experiments did not work or produced confounding results. Being a father to Kaya and Nathan is as much, if not more, a part of my identity as being a scientist. I love you both very much. Finally I would like to thank Jen for catching the last few typos I had in this thesis. iv TABLE OF CONTENTS Abstract: ii Acknowledgments: iv Chapter 1: Introduction 1 I: Epigenetics of Ciliates 1.Ciliate Biology 2 2. Epigenetic Phenomena in Ciliates 12 3. RNA-Mediated Epigenetic Mechanisms 20 4. Small RNA-Mediated DNA Rearrangements 25 5. Chromosome Fragmentation and Elimination of DNA During 50 During Conjugation in Oxytricha II: Double-Stranded RNA Binding Motif Proteins 53 III: Scope of the Thesis 55 Chapter 2: Transcription of dsRNA from the R IES is Required for 86 Subsequent Targeting and DNA Elimination Late in Conjugation in Tetrahymena thermophila Chapter 3: Germ Line Transcripts are Processed by a Dicer-Like Protein 106 that is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila Chapter 4: The Role of the Tandem Double-Stranded RNA Binding Motif 123 Protein, DRB2, in DNA Elimination in the Ciliate, Tetrahymena thermophila Chapter 5: The Double-Stranded RNA Binding Motif Protein, DRB1, 173 Plays an Auxiliary Role in Meiosis I in the Ciliate, Tetrahymena thermophila Chapter 6: Future Perspectives 207 Appendix: Subtraction by Addition: Domesticated Transposases in 221 Programmed DNA Elimination v FIGURES AND TABLES Chapter 1: Introduction Page Figure 1: Ciliate life cycle 74 Figure 2: Somatic genome rearrangements of ciliates through chromosome 75 fragmentation and DNA elimination Table 1: Histone modifications found in the nuclei of ciliates 76 Figure 3: Cytoplasmic inheritance-mating type determination in 77 Paramecium Figure 4: Epigenetic inheritance of mating type in Paramecium 78 demonstrated by the mutation of mtFE Figure 5: RNAi-dependent DNA elimination in P. tetraurelia 79 Figure 6: RNAi and heterochromatin components of RNAi-dependent 81 DNA elimination in T. thermophila Figure 7: Histone methylation triggers DNA elimination of internal 83 eliminated sequences (IESs) in T. thermophila Figure 8: Template-guided gene unscrambling, DNA elimination and 85 chromosome fragmentation in O. trifallax Chapter 2: Transcription of dsRNA from the R IES is Required for Subsequent Targeting and DNA Elimination Late in Conjugation in Tetrahymena thermophila Figure 1: Diagram of R IES micro- and macronuclear loci in wild-type 100 and R IES knockout strains Figure 2: Creation and verification of R IES knockout strains 101 Table 1: Progeny production of R IES mic knockouts in wild-type and 102 knockout matings Figure 3: Failure of plasmid-based R IES rearrangement in homozygous 103 micronuclear R IES knockout strain matings Table 2: Oligonucleotides used in the course of this study 105 Chapter 3: Germ Line Transcripts are Processed by a Dicer-Like Protein that is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila Table 1: Oligonucleotides used in course of this study 111 Figure 1: Tetrahymena thermophila encodes three Dicer-like proteins 113 Figure 2: Germ line knockout of DCL1 114 Figure 3: ΔDCL1 strains arrest late in conjugation 115 Figure 4: Conjugating ΔDCL1 strains exhibit loss of small RNA 116 production and germ line transcript accumulation Figure 5: The DCL1 protein is localized to meiotic micronuclei. 117 Figure 6: DCL1 knockouts fail to excise micronucleus-limited DNA 118 Figure 7: Chromosome breakage does not occur in DCL1 knockouts 118 vi Page Figure 8: DCL1 is not required for H3K9 methylation 119 Chapter 4: The Role of the Tandem Double-Stranded RNA Binding Motif Protein, DRB2, in DNA Elimination in the Ciliate, Tetrahymena thermophila Figure 1: T. thermophila contains two predicted tandem double-stranded 150 RNA binding motif proteins Figure S1: ClustalW alignment of dsRNA binding motifs (DSRMs) of D. 151 melanogaster Loqs and R2D2, C. elegans RDE-4, H. sapiens TRBP2 and T. thermophila DRB2 and DRB1 Figure S2: Alignment of DRB1 and DRB2 protein sequences outside
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