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DISCOVERY and CHARACTERIZATION of PATHWAYS INVOLVED in FUS and TDP43-INDUCED TOXICITY in YEAST a Thesis Submitted in Partial

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DISCOVERY and CHARACTERIZATION of PATHWAYS INVOLVED in FUS and TDP43-INDUCED TOXICITY in YEAST a Thesis Submitted in Partial DISCOVERY AND CHARACTERIZATION OF PATHWAYS INVOLVED IN FUS AND TDP43-INDUCED TOXICITY IN YEAST A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science By WESTON JOSEPH SHAW B.S., Wright State University, 2017 2020 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL DATE OF DEFENSE 04 / 30 /2020 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY WESTON JOSEPH SHAW ENTITLED DISCOVERY AND CHARACTERIZATION OF FUS AND TDP43-INDUCED TOXICITY IN YEAST BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE. Shulin Ju, Ph.D. Thesis Director Eric Bennett, Ph.D. Chair, Department of Neuroscience, Cell Biology and Physiology Committee on Final Examination Chris Wyatt, Ph.D. Thomas Brown, Ph.D. Barry Milligan, Ph.D. Interim Dean of the Graduate School ABSTRACT ShaW, Weston Joseph. M.S., Department of Neuroscience, Cell Biology, and Physiology, Wright State University, 2020. Discovery and characterization of pathways involved in FUS and TDP43-induced toxicity in yeast. High-throughput genome-scale studies are becoming increasingly common as a means to discover genetic interactions. This methodology is particularly efficient When performed in the budding yeast Saccharomyces cerevisiae. Here, We (1) overexpress a large human gene library in yeast to assess how many of them are toxic, and (2) use the list of genes generated above to refine and analyze human genes previously identified to enhance toxicity of tWo ALS-associated proteins, FUS and TDP-43. By introducing each of 13,500 human genes into yeast, we demonstrated that the majority of these genes (about 97%) are not toxic to yeast When overexpressed. These results indicated that toxicity of human genes, such as FUS and TDP-43, are very likely due to their interactions With specific cellular pathways, rather than simply a non-specific effect of their overexpression in yeast. This is supported by our analysis showing that the toxic human genes are enriched in RNA metabolic processes and DNA transcription, both of Which are conserved betWeen yeast and human. Our lab previously identified a preliminary list of 685 human genes that enhance toxicity of FUS and TDP-43 in yeast. However, none of these genes Were tested for their own toxicity, so they Were possibly false positives, and never Were removed from the list. Using the toxic genes identified above, we refined the enhancers list from 685 to 358 genes, out of Which 138 genes enhanced TDP-43 toxicity, 335 genes enhanced FUS toxicity, and 115 genes enhanced both. Interestingly, functional classification of the genes that enhance toxicity from both FUS and TDP-43 revealed a group of cell cycle iii regulators that have been linked to the DNA damage response (DDR) and repair. Given that FUS and TDP-43 are RNA-binding proteins that have been implicated in DDR, these results suggest a possible mechanism of FUS and TDP-43 toxicity involving their abnormal activation of the cell cycle. iv TABLE OF CONTENTS Page I. INTRODUCTION ................................................................................................ 1 Amyotrophic Lateral Sclerosis ..................................................................... 1 Epidemiology ................................................................................................ 3 The Genetics Of ALS .................................................................................... 4 The Role of TDP-43 In ALS ......................................................................... 7 The Role of FUS In ALS .............................................................................. 10 The Cell Cycle And ALS .............................................................................. 12 A Yeast Model of FUS And TDP-43 ............................................................ 15 Overexpression A Human Gene Library In Yeast ........................................ 18 Hypothesis ..................................................................................................... 19 Summary ...................................................................................................... 19 II. MATERIALS AND METHODS ........................................................................ 21 Yeast Strains And Media .............................................................................. 21 Human Gene Library .................................................................................... 21 Screening for Human Genes Toxic To Yeast ............................................... 23 Yeast Mating To Bring Plasmids Together .................................................. 25 Serial Dilution Growth Assay ...................................................................... 25 Fluorescence Microscopy ............................................................................. 25 Western Blot ................................................................................................ 26 Gene Ontology Enrichment Analysis .......................................................... 27 III. RESULTS .......................................................................................................... 28 v Genetic Screen For Toxic Human Genes In Yeast ....................................... 28 Enrichment Analysis Of Toxic Human Genes .............................................. 31 Refining A Set Of FUS And TDP-43 Enhancers .......................................... 37 Confirming FUS And TDP-43 Enhancers .................................................... 38 Enrichment Analysis Of FUS And TDP-43 Enhancers ................................ 40 Cell Cycle Regulators Enhance FUS And TDP-43 Toxicity In Yeast ........ 43 Hydroxyurea Enhances FUS and TDP-43 Induced Toxicity ........................ 45 Cell Cycle Regulators Do NOT Change FUS and TDP-43 Aggregation Or Localization .......................................................................................... 47 Cell Cycle Regulators Do NOT Affect FUS and TDP-43 Protein Level ..... 47 IV. DISCUSSION .................................................................................................... 50 Screening For Human Genes Toxic To Yeast .............................................. 50 Analyzing a set of FUS and TDP-43 enhancers ........................................... 52 Future experiments ....................................................................................... 52 Conclusion .................................................................................................... 55 V. REFERENCES .................................................................................................... 56 vi LIST OF FIGURES Figure Page 1. Motor Neuron Death And Muscle Atrophy In ALS ........................................... 2 2. ALS Gene Discovery By Year ............................................................................ 6 3. The RNA Recognition Motifs (RRMs) of TDP-43 Mediate Many Steps Of RNA Processing ........................................................................................................... 9 4. Similar To TDP-43, The RRM Of FUS Mediates Many Steps of RNA Processing ............................................................................................................................. 11 5. Schematic Of The Cell Cycle Phases And The Corresponding Phase-Specific Promoting Factors ............................................................................................... 13 6. The Overexpression Of Wild-type (WT) FUS And TDP-43 In Yeast Reduces Yeast Cell Fitness And Produces Cytosolic Inclusions ...................................... 16 7. Plasmid Map Of pRS416Gal1-ccdB ................................................................... 24 8. Mating-based Overexpression Screening of A Human Gene Library ................ 29 9. The Representative Glucose (Top) And Galactose Agar Plates (Bottom) From The Human Gene Library Screen For Toxic Human Genes ...................................... 30 10. ~3% Human Genes Are Toxic To Yeast ............................................................ 32 11. Distribution Profile Of FUS And TDP-43 Enhancers ........................................ 39 12. Cell Cycle Regulators Enhance FUS (A) And TDP-43 Induced Toxicity (B) .. 44 13. 10mM Hydroxyurea Enhances FUS (A) And TDP-43 (B) Induced Toxicity in Yeast ................................................................................................................... 46 14. CDK2 Or CKS1 Does Not Alter FUS Or TDP-43 Aggregate Quantity And Localization ......................................................................................................... 48 vii 15. FUS And TDP-43 Protein Level Does Not Change When Co-expressed With CDK2 And CKS1 .......................................................................................................... 49 16. Yeast Cellular And Nuclear Morphology Changes During Progression Through The Stages Of The Cell Cycle .................................................................................... 54 viii LIST OF TABLES Table Page 1. List of Yeast Strains Used In This Study ............................................................ 22 2. Gene Term Analysis For Toxic Human Genes .................................................. 33 3. Enrichment Analysis For GO Biological Process Terms Among The Human Genetics Enhancers Of TDP-43 Induced Toxicity in Yeast ............................... 41 ix ACKNOWLEDGEMENTS For their support and
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