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MECHANISMS of HUMAN LARP6 NUCLEAR EXPORT by Samantha Kathleen Zepeda, B.S. a Thesis Submitted to the Graduate Council of Texas S

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MECHANISMS of HUMAN LARP6 NUCLEAR EXPORT by Samantha Kathleen Zepeda, B.S. a Thesis Submitted to the Graduate Council of Texas S MECHANISMS OF HUMAN LARP6 NUCLEAR EXPORT by Samantha Kathleen Zepeda, B.S. A thesis submitted to the Graduate Council of Texas State University in partial fulfillment of the requirements for the degree of Master of Science with a Major in Biochemistry May 2019 Committee Members: Karen A. Lewis, Chair Tania Betancourt Wendi David COPYRIGHT by Samantha Kathleen Zepeda 2019 FAIR USE AND AUTHOR’S PERMISSION STATEMENT Fair Use This work is protected by the Copyright Laws of the United States (Public Law 94-553, section 107). Consistent with fair use as defined in the Copyright Laws, brief quotations from this material are allowed with proper acknowledgement. Use of this material for financial gain without the author’s express written permission is not allowed. Duplication Permission As the copyright holder of this work I, Samantha Kathleen Zepeda, authorize duplication of this work, in whole or in part, for educational or scholarly purposes only. ACKNOWLEDGEMENTS Science is not an independent pursuit and for this reason, I would like to acknowledge the many people in my life who have assisted me on this journey. In my personal life, I would like to thank my friends and family for their support. In particular, I would like to thank my husband for his support, patience and endless coffee runs. Additionally, my siblings, Jay and Kaitlynn Griesenbeck, who have always given me motivation to continue reaching for the next challenge. Professionally and personally, my mentor, Dr. Karen A. Lewis, has been a wonderful guide on this journey and I would like to thank her for all of the time, support and mentorship that she has provided. In her lab, I have had the opportunity to fall in love with research and grow into a more knowledgeable and proficient researcher. Her lab has changed my career path and set me on the road to a Ph.D. program. I originally believed that the path I am now on, was unattainable, and I will forever be grateful for the role Dr. Lewis has provided in helping me develop the skills and confidence to continue on. My lab mates past and present have been invaluable resources to me throughout my time here. In particular I would like to thank Ms. Leticia Gonzalez for her contribution of Ran-GTP for this project and the initial cloning and purification attempts for CRM1 and SUMO full-length LARP6. Her presence, advice, and preparation of reagents and maintenance of cell culture lines has been invaluable. iv I would also like to thank Melissa Carrizales for her kindness and feedback. She has been my sounding board throughout this journey and I am forever grateful for her attitude, intelligence, and friendship. Julia Roberts and Chelsea Toner have provided endless feedback and support. I would also like to thank José Castro and Eliana Pena for their training and guidance when I started this journey. In addition, I would also like to thank Dr. Joe Koke, Dr. Raquel Salinas, and Dr. Tania Betancourt for their advice and support as we set up a cell culture room for my project. Without their guidance I would truly be lost. Lastly, I would like to thank the Texas State University Analytical Research Service Center (ARSC) and its staff for their technical expertise, training, and guidance in cellular imaging. I would also like to thank Alissa Savage in particular for her endless patience, guidance, and support in cellular imaging and cell culture. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .................................................................................... iv LIST OF TABLES ............................................................................................... vii LIST OF FIGURES ..............................................................................................viii LIST OF ABBREVIATIONS ................................................................................. xii ABSTRACT .........................................................................................................xiii CHAPTER I. INTRODUCTION ........................................................................................ 1 II. MATERIALS AND METHODS .................................................................. 19 III. IS LARP6 AN IN VIVO TARGET FOR CRM1 MEDIATED NUCLEAR EXPORT?............................................................................... 50 IV. IN VITRO INTERACTION BETWEEN LARP6 AND CRM1 ...................... 70 V. DELETING PUTATIVE NES IN RECOMBINANT LARP6 ...................... 109 VI. DISCUSSION ......................................................................................... 113 APPENDIX SECTION ....................................................................................... 117 REFERENCES.................................................................................................. 125 vi LIST OF TABLES Table Page 1. Plasmids and Antibiotic Resistance ............................................................... 20 2. E. coli Cell Lines and Antibiotic Resistance ................................................... 20 3. Size Exclusion Chromatography S75 Standards symbols ............................. 26 4. Size Exclusion Chromatography S200 Standards symbols ........................... 27 5. Buffers for batch purification and SEC of GST-NES peptides........................ 31 6. Buffers for batch purification and SEC of GST-NES peptides with 5% Glycerol ............................................................................................ 31 7. HsCRM1 Purification Buffers ......................................................................... 36 8. Ran-GTP Purification Buffers......................................................................... 39 9. Buffers for Nickel Affinity Purification and SEC of Sumo-LARP6 ................... 42 10. Human Cell Culture Lines ............................................................................. 44 11. LocNES-identified NES ................................................................................. 71 12. Random Sequence Generation ..................................................................... 87 13. Broth Composition Differences for Scaled Expressions ............................... 103 14. OE-PCR fragment identifier and expected molecular weight ....................... 109 vii LIST OF FIGURES Figure Page 1. Topology of the La Related Protein Superfamily .............................................. 3 2. Solution NMR structures of human LARP6 ...................................................... 5 3. Mechanism of CRM1 mediated nuclear export ................................................ 7 4. Crystal structure of CRM1/Snurportin-1 Complex ............................................ 9 5. Nuclear Export Motifs: Consensus Sequences .............................................. 10 6. Altered Localization of GFP-LARP6 mutants in vivo ...................................... 14 7. Location of putative localization motifs within the LARP6 RRM ..................... 15 8. Size Exclusion Chromatography S75 Standardization Curve. ....................... 26 9. Size Exclusion Chromatography S200 Standardization Curve ...................... 27 10. Cell Lysate Screening with commercial anti-LARP6 antibody ........................ 52 11. HEK-293 Seeding Density and Adherence on Poly-D-Lysine coverslips ....... 53 12. Fixation and antibody dilution screening HEK-293 P16 ................................. 56 13. LMB inhibition of CRM1 screening in HEK-293 cells ..................................... 58 14.Nonspecific antibody signal in HEK-293 P29 cells with secondary antibody (1:500) ................................................................................. 61 15. Nonspecific antibody signal in HEK-293 P34 and P37 cells with secondary antibody (1:1000) ....................................................................... 61 16.LMB inhibition of CRM1 screening in MCF-7 cells ......................................... 63 17.Cell Lysate Screening maximal protein concentrations .................................. 65 18.Cell lysate screening with Cell lysate Method 2 ............................................. 66 viii 19.Human Protein Atlas LARP6 RNA expression by cell line ............................. 68 20. LocNES-identified RRM putative NES .......................................................... 72 21.Cartoon depiction of NES bait interaction/pull down mechanism ................... 73 22.Annealing of Putative NES Oligonucleotides.................................................. 74 23.Annealing of Experimentally confirmed NES Oligonucleotides ...................... 75 24.Double Digest of pGEX4T3-TEV plasmid with EcoRI and XhoI Endonucleases .................................................................................... 76 25.Preparative Expression of GST-LARP6(179-193) .......................................... 78 26.Preparative Expression of GST-LARP6(210-224) .......................................... 79 27.Preparative Expression of GST-LARP6(457-471) .......................................... 80 28.Preparative Expression of GST-HIVrev (Class II NES) .................................. 81 29.Preparative Expression of GST tag ................................................................ 82 30.Batch Purification of GST tag ......................................................................... 83 31.Batch Purification of GST-HIVrev ................................................................... 84 32.Batch Purification of GST tagged LARP6(457-471) ......................................
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