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Quality Control Under Stress: Ubiquilin 2 Bridges Protein and Rna Homeostasis at Stress Granules in Als/Ftd Pathology

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Quality Control Under Stress: Ubiquilin 2 Bridges Protein and Rna Homeostasis at Stress Granules in Als/Ftd Pathology QUALITY CONTROL UNDER STRESS: UBIQUILIN 2 BRIDGES PROTEIN AND RNA HOMEOSTASIS AT STRESS GRANULES IN ALS/FTD PATHOLOGY by Elizabeth J. Alexander A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland January 2019 © 2019 Elizabeth J. Alexander All Rights Reserved Abstract Both protein and RNA quality control are critical for cell survival during stress and are disrupted in the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is the most common adult onset motor neuron disease, while FTD is the second most common type of dementia for individuals under 65. Both are characterized by the accumulation of proteins in intracellular inclusions and are thought to be part of a continuous clinical spectrum, but their causes are largely unknown. Stress granules (SGs), a type of RNA granule, have recently emerged as potential seed sites for patient protein inclusions that incorporate both protein quality control (PQC) factors and RNA binding proteins (RBPs). SGs transiently sequester stalled translation initiation complexes and apoptosis signaling molecules in response to stress. These dynamic particles form via a liquid-liquid phase separation mediated by oligomerization of the low complexity prion-like domains in RBPs bound to RNA in a process tuned by RNA length and structure. Stable interactions between these proteins in SGs leads to the formation of protein aggregates. The PQC factors that regulate the dynamic incorporation of RBPs into SGs to prevent aggregate formation remain largely unknown. In 2011, missense mutations in the ubiquitin-like protein ubiquilin 2 (UBQLN2) were found to cause ALS with FTD in rare X-linked cases. In an effort to characterize UBQLN2’s normal cellular functions, I performed a proteomic screen for UBQLN2 interactors and found an enrichment of RBPs including SG components under non-stress conditions. Using an unconventional staining technique, I confirmed that UBQLN2 localizes to SGs under a variety of stress conditions and found that this association is transient. In the time period that UBQLN2 localizes to SGs, it also suppresses large SG formation suggesting that it modulates incorporation of SG components. To characterize UBQLN2’s regulatory role in SG formation, I focused on its interaction with the RBP fused in sarcoma (FUS). FUS was not only the most enriched RBP among the UBQLN2 interactors, but also is genetically and pathologically linked to ALS/FTD. In a single molecule Förster resonance energy transfer (FRET) assay, UBQLN2 increased the dynamics of FUS-RNA interaction, increasing the fluidity of FUS-RNA complex formation. This effect translated to a decrease in FUS partitioning ii into liquid droplets in vitro and suppression of FUS-GFP recruitment into SGs in cells suggesting that UBQLN2 slows FUS recruitment into SGs by increasing the dynamics of its interaction with RNA. ALS/FTD-linked mutations in UBQLN2 partially impaired UBQLN2’s ability to increase the dynamics of FUS-RNA interaction and suppress SG formation, indicating a partial loss of UBQLN2 function. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid-liquid phase separation of RBPs in SG formation by affecting the dynamics of RNA-protein interaction. Because SGs potentially seed toxic ALS/FTD patient inclusions, these findings have implications for understanding ALS/FTD pathogenesis and designing new treatments for these diseases. Thesis advisor: Dr. Jiou Wang Thesis readers: Dr. Jiou Wang, Dr. Michael J. Matunis, Dr. Susan Michaelis, Dr. Marie Hardwick Alternate thesis readers: Dr. Anthony Leung, Dr. Mark Kohr iii Acknowledgements My thesis project has been a long and winding journey from worm genetics, to cell-based autophagy assays, to DNA damage repair assays and finally stress granule studies, but it has been enjoyable thanks to the support of many friends, family and colleagues. I am truly grateful to Dr. Jiou Wang and the BMB department for their support and the opportunity to pursue these studies at Johns Hopkins. Hopkins is a place where there seem to be an endless number of opportunities to exchange knowledge and form new collaborations. I will miss the School of Public Health’s spirit of inclusion where in a single day I will receive an invitation to attend an African Public Health network symposium, STD journal club, and biostatistics help session. This has truly been a great place to spend the past seven years. Were it not for the time and generosity of many professors, staff, postdocs and students, my thesis project would not have been possible. First, I would like to thank Dr. Jiou Wang for taking me into the lab as his first PhD student and helping me to always try for something slightly out of reach. I greatly appreciate his optimism, idealism and drive to achieve great things and am grateful to him for his patience with the development of the ubiquilin 2 project. I would like to thank all of the past and present Wang lab members and many BMB students who so unselfishly offered their time and technical advice during my PhD, especially Dr. Goran Periz, a master at building new genetic tools who taught me everything I know about molecular cloning, Dr. Yen-ching Wu who taught me the meaning of good “controls”, Dr. Aaron Haeusler with Peter Steinwald who helped me to purify the UBQLN2 protein, and Dr. Janet Ugolino, who has been a friend and colleague always willing to help me troubleshoot and without whom I would not have remained sane while writing this thesis. Also, a special thanks to two collaborators, Dr. Raja Sekhar who put up with my persistent requests to process mass spec samples in the Pandey lab and Dr. Jaya Sarkar in Sua Myong’s lab who did the initial single molecule FRET experiments with purified FUS, UBQLN2, and RNA to show that UBQLN2 increases FUS-bound RNA dynamics. iv I also greatly appreciate the help and guidance from many of the BMB faculty members, especially Dr. Mike Matunis who always seems to find the time to provide thoughtful advice, Dr. Val Culotta who has given me feedback on my project since the beginning in joint lab meetings and whom I greatly admire, and Dr. Fengyi Wan who always manages to give me some encouraging words. Many thanks also to my thesis committee members Dr. Susan Michaelis, Dr. Anthony Leung, and Dr. Marie Hardwick for their advice and support through the years. Also, thanks to the students and postdocs in BMB never too busy to stop to chat. To the guys of the Leung Lab, Lyle, Aravinth, and Mohsen, for the many fun discussions about stress, RNA granules and science in general. To my BMB classmates Nicole, Chynna, Fengrong and Xin for many fun nights out to celebrate our milestones in the PhD program together. To my late-night companions Marina and Ho-yon for keeping me company. And to a few of my senior classmates Katherine Reiter, Jolyn Gisselberg, Eric Wier and Casey Daniels who have offered help and advice over the years. I probably would not have come to JHSPH had it not been for the support of my undergraduate advisor at Indiana University, Dr. Lingling Chen, my supervisor at the University of Wisconsin, Dr. Susan West, my manager at Nektar Therapeutics Shaoyong Ni, and Dr. Carmen Scholz, at the University of Alabama, Huntsville who supported my effort to return to graduate school in biochemistry after many years of working in industry as an analytical chemist. I am also grateful to Ms. Sharon Warner who convinced me to write another essay to the admissions committee about my commitment to a five-year PhD program and supported me during my first years at Hopkins. Many thanks to the BMB staff who kept the department running smoothly, especially Ms. Shannon Gaston who helped me in innumerable ways in preparing for my thesis defense. Lastly, thanks to my family. First, to my Mom and Dad, Jean and Rich Alexander for always being there to listen and sacrificing so many mornings, noons and nights for my many years in school. To my Mom who gave me the tools to be a confident, assertive woman and my Dad who taught me to negotiate in life. They have always been there when no one else would be. I could not be luckier. To v my sister Sarah for always reminding me what is important in life and for being one of the dancers rather than one of the skeptics. And to my husband Chris who has been my champion and friend from preparing late night dinners to putting up with the inevitable 30-minute lab job turning into 3 hours, who has spent hours upon hours in rush hour Beltway traffic to pick up our daughter from daycare so that I could spend a few more hours working every night and never complains. And last but not least, to my daughter Sophie who always reminds me to be stronger, faster and more prompt for whom I hope to be the best Mom-scientist possible. vi Table of Contents Abstract ................................................................................................................................................................. ii Acknowledgements ............................................................................................................................................. iv Table of Contents .............................................................................................................................................. vii List of Tables ......................................................................................................................................................
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