UC Riverside UC Riverside Electronic Theses and Dissertations Title Characterization of the DHH1/DDX6-Like RNA Helicase Family in Arabidopsis thaliana Permalink https://escholarship.org/uc/item/09112483 Author Chantarachot, Thanin Publication Date 2018 Supplemental Material https://escholarship.org/uc/item/09112483#supplemental License https://creativecommons.org/licenses/by-nc-nd/4.0/ 4.0 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE Characterization of the DHH1/DDX6-Like RNA Helicase Family in Arabidopsis thaliana A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Genetics, Genomics and Bioinformatics by Thanin Chantarachot December 2018 Dissertation Committee: Dr. Julia Bailey-Serres, Chairperson Dr. Xuemei Chen Dr. Fedor Karginov Copyright by Thanin Chantarachot 2018 The Dissertation of Thanin Chantarachot is approved: Committee Chairperson University of California, Riverside Acknowledgements I would like to express my sincere gratitude to my advisor, Dr. Julia Bailey- Serres, for welcoming me into her group. Without her patience, support, and insightful guidance, my PhD studies would not have been possible. To me, she is not only my advisor, but also a role model. I could not ask for a better mentor. I will cherish the memory of her wonderful mentorship and pass this on to my future students. I would also like to thank the other members of my dissertation committee, Dr. Xuemei Chen and Dr. Fedor Karginov, for their helpful comments and suggestions on my dissertation project. I thank the faculty who served on my guidance and qualifying exam committees: Dr. Thomas Eulgem, Dr. Joel Sachs, Dr. Amy Litt, and Dr. Natasha Raikhel. I thank current and former members of the Bailey-Serres lab for sharing with me their critical comments, encouragement, and ideas. Particularly, I thank Dr. Reed Sorenson who initiated this project and mentored me during the first two years of my PhD studies. I thank Dr. Maureen Hummel for her teaching and contribution on RNA-seq library preparation and analysis. I thank Dr. Jeremie Bazin, Dr. Mauricio Reynoso, Dr. Germain Pauluzzi, Dr. Anna Locke, Dr. Matthew Prior, Dr. Miguel Lopez, Dr. Erin Brinton, Dr. Rejbana Alam, Dr. Travis Lee, Dr. Lauren Dedow, Sonja Winte, and ALek Kettenburg for their help and support. I also thank Dr. Dawn Nagel and her group members, Dr. Titouan Bonnot, Emily Blair, and Tejasvinee Mody, for helpful comments on this project as well as encouragement. I thank everyone at UCR that has helped me throughout my PhD studies: Dr. David Carter for his guidance and assistance with microscopy; Dr. Haiyan Ke for her iv help with SA quantification; and members of the Dehesh and Chen lab for sharing ideas and reagents when needed. I thank Dr. Leslie Sieburth for her guidance and technical support on the mRNA decay experiment during my visit to her lab at the University of Utah. I thank Alba González Hernando, Dr. Moritz Leber, and Dr. Malia Deshotel for their friendship during my time in Utah. I thank my former mentors Dr. Upatham Meesawat and Prof. Choathip Purintavaragul at Prince of Songkla University, as well as Dr. Supachitra Chadchawan and Dr. Teerapong Buaboocha at Chulalongkorn University for laying the foundation and helping pave the way for my scientific career. It was indeed Dr. Meesawat’s pigeon orchid that lured me into plant research, and in Dr. Chadchawan’s lab that I fell in love with plant molecular biology. I thank the Thailand’s Institute for the Promotion of Teaching Science and Technology (IPST) for its full-scholarship through the Development and Promotion of Science and Technology Talents Project throughout 12 years of my educational career. Without its financial support, it would not have been possible for someone from my socioeconomic background to achieve highest education in the United States as I did today. This scholarship also provided me with wonderful mentors and friends that I am thankful for to the rest of my life. I am grateful to the administrative personnel at IPST and the Royal Thai embassy that has helped and taken care of me with kindness throughout this journey. I thank my friends from college Dr. Supattra Thepnarong and Dr. Lompong Klinnawee for their continued friendship and moral support. I thank Bhanubhakta Kaan Jittiang for being there for me during my tough time. Thank you for being by people! v I thank my Royal Thai fellows and friends around the world for sending kind words and their friendship during this invaluable journey. I thank the Thai community and friends for their friendship and support: Dr. Yoothana Thanmongkhon, Dr. Supawadee Wichitchan, Dr. Parawee Pumwongpitak, Dr. Panu Suppatkul, Wipawee Leesutthiphonchai, and Nuttapol Pombubpa. Finally, I thank my parents and my grandmother for their unconditional love and support. My parents did not have a chance to finish high school, but they have worked extremely hard to provide me and my brother and sister with the best educational opportunity they could possibly provide. Without their sacrifice, I would not have had this achievement. I thank my brother and sister, Joke and June, for being such wonderful siblings. I am lucky to be their brother. Publications Chantarachot T, Bailey-Serres J (2018) Polysomes, Stress Granules, and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function. Plant Physiol 176: 254–269 vi ABSTRACT OF THE DISSERTATION Characterization of the DHH1/DDX6-Like RNA Helicase Family in Arabidopsis thaliana by Thanin Chantarachot Doctor of Philosophy, Graduate Program in Genetics, Genomics and Bioinformatics University of California, Riverside, December 2018 Dr. Julia Bailey-Serres, Chairperson General cytoplasmic mRNA turnover in eukaryotes is governed by two major pathways: 5’-to-3’ decay initiated by the multi-subunit mRNA decapping complex and carried out by EXORIBONUCLEASE 1/4 and 3’-to-5’ decay executed by the RNA exosome or the 3’-to- 5’ exoribonuclease SUPPRESSOR OF VARICOSE (SOV). In yeast and metazoa, DEAD- box RNA helicase DHH1/DDX6 proteins are associated with the decapping complex and involved in translational repression and/or mRNA degradation. In this dissertation, RNA HELICASE 6, 8 and 12 (RH6, RH8 and RH12) were identified as orthologs of the DHH1/DDX6 family in the plant Arabidopsis thaliana. Genetic analysis uncovered that the genes RH6, RH8 and RH12 are functionally redundant and indispensable for growth and development, as the triple homozygous rh6 rh8 rh12 mutant displays pleiotropic phenotypes including delayed germination, altered vein patterning, and severe maturation defects including infertility. By use of confocal microscopy, we found that all the three RHs are nucleocytoplasmic proteins, with the cytoplasmic portion forming stress-inducible vii cytoplasmic ribonucleoprotein complexes associated with both processing bodies (PBs) and stress granules (SGs). Evaluation of PB and SGs in the triple homozygous mutant rh6 rh8 rh12 indicated that the RHs are required for PB but not SG formation. Genome- wide analysis of RNA decay kinetics in the rh6 rh8 rh12 mutant by genome-scale RNA- sequencing revealed that the RHs mediate degradation of short-lived mRNAs with diverse biological functions, the majority of which are putative substrates of the 5’ to 3’ decapping- mediated decay pathway. Further genome-scale polyadenylated mRNA and polyadenylated ribosome-associated mRNA analysis revealed that co-disruption of these RHs perturbed transcriptome and translatome homeostasis, characterized by an increase in the steady-state mRNA abundance and translational status of stress- and defense- response transcripts and the simultaneous repression in accumulation of growth and development-associated transcripts. Genetic analysis determined that the growth defects of rh6 rh8 rh12 are not related to small interfering RNA production, but caused by a constitutive immune response linked with stabilization and enhanced ribosome occupancy of defense-related mRNAs. The phenotypes, constitutive elevation of stress/defense- response mRNAs and repression of growth-related mRNAs was alleviated by (over)expression of a functional SOV allele. This study determined the plant DDH1/DDX6 orthologs are a variable component of the 5’ to 3’ decay machinery that accelerates decay of growth limiting defense-associated transcripts under non-stress conditions. viii TABLE OF CONTENTS Chapter 1 Polysomes, Stress Granules and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function……. 1 1.1 ADVANCES…………..……………..………………………..………………..………. 1 1.2 INTRODUCTION……..……………..……………..……………..…………………… 1 1.3 CONNECTIONS BETWEEN mRNA DECAY AND TRANSLATION……..………. 2 1.3.1 mRNA surveillance-triggered decay………..…….……………..………........ 3 1.3.2 XRN4-mediated co-translational decay……..………………..……………… 4 1.3.3 miRNA-triggered decay of translating mRNAs……..………………..……… 6 1.4 GENERAL CYTOPLASMIC mRNA DEGRADATION……..………………..……... 8 1.4.1 Deadenylation……..………………..………………..………………..……….. 9 1.4.2 Decapping……..………………..………………..………………..…………… 11 1.4.3 5’-3’ degradation……..………………..………………..………………..……. 15 1.4.4 3’-5’ degradation……..………………..………………..………………..……. 15 1.5 mRNA SEQUESTRATION IN STRESS GRANULES – A TRANSIT DEPOT BETWEEN TRANSLATION, STORAGE AND DECAY?...................................... 17 1.6 HETEROGENEOUS MACROMOLECULAR HUBs OF CYTOPLASMIC mRNA TRANSLATION, TURNOVER AND SEQUESTRATION……..………………..….