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Post-Transcriptional Regulation Of POST-TRANSCRIPTIONAL REGULATION OF SELENOPROTEIN S by ERIC MICHAEL COCKMAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine CASE WESTERN RESERVE UNIVERSITY August, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Eric Cockman Candidate for the degree of Doctor of Philosophy.* Donal Luse, Ph.D. Committee Chair Donna Driscoll, Ph.D. Research Advisor and Committee Member Richard Padgett, Ph.D. Committee Member Ofer Reizes, Ph.D. Committee Member Bela Anand-Apte, M.D., Ph.D. Committee Member Date of Defense June 28, 2019 *We also certify that written approval has been obtained for any proprietary materials contained therein. i Table of Contents List of Tables................................................................................................. iv List of Figures................................................................................................. v Acknowledgements...................................................................................... vii Abstract........................................................................................................... x CHAPTER I: INTRODUCTION.................................................................... 1 Selenium and Selenocysteine...................................................................... 1 Synthesis of Sec...................................................................................... 2 The genetic code..................................................................................... 5 An overview of general translation elongation and termination........................ 7 Insertion of Sec into a growing polypeptide.................................................. 11 Regulation of Sec insertion into selenoproteins............................................. 16 Selenoproteins in the human body.............................................................. 19 Selenoprotein S...................................................................................... 23 SelS in disease........................................................................................ 26 CHAPTER II: MATERIALS AND METHODOLOGY.............................. 30 CHAPTER III: RESULTS............................................................................ 40 Recoding efficiency of the SelS 3’ UTR...................................................... 40 V5-surrogate assay for Sec insertion........................................................... 42 Sec is inserted into the SelS-V5 surrogate assay........................................... 44 ii Efficiency of the SelS-V5 reporter............................................................. 48 Distance restriction of SelS recoding.......................................................... 52 The proximal SelS 3’ UTR contains two distinct conserved sequences............. 55 PSL and SPUR mutations do not affect RNA levels or general translation......... 60 PSL and SPUR mutation effects UGA recoding are not cell-type specific......... 62 Point mutations in the SPUR Element inhibit UGA recoding........................... 64 The SPUR element does not function with other SECIS elements.................... 67 Deletion of the PSL promotes Sec insertion in SelS-V5.................................. 69 Other sequences can functionally replace the SelS PSL.................................. 73 The SPUR is required for efficient Sec insertion in the FLAG-SelS.................. 79 The effect of the SPUR element is position dependent.................................... 84 CHAPTER IV: DISCUSSION AND FUTURE DIRECTIONS................... 87 APPENDIX................................................................................................ 103 REFERENCES CITED.............................................................................. 104 iii List of Tables Table 1: List of accession numbers..................................................................... 30 Table 2: List of mutagenic primers.......................................................................... 32 Table 3: Primers used for insertion PCR............................................................... 33 Table 4: Primers used for qRT-PCR...................................................................... 36 Table 5: Recoding efficiencies of 3’UTRs............................................................ 41 Table 6: List of peptides queried for SelS-V5 terminal PRM.................................... 47 Table 7: Selenoprotein S internal peptides.............................................................. 72 Table 8: List of peptides queried for FLAG-SelS PRM........................................... 83 iv List of Figures Figure 1: Synthesis of Selenocysteine............................................................................... 4 Figure 2: The genetic code................................................................................................ 6 Figure 3: Translational elongation.................................................................................... 9 Figure 4: Translational termination................................................................................ 10 Figure 5: The SECIS element......................................................................................... 14 Figure 6: Model of UGA recoding.................................................................................. 15 Figure 7: Recoding efficiencies of SECIS elements from different selenoproteins........ 18 Figure 8: The Human selenoproteome............................................................................ 22 Figure 9: Selenoprotein S............................................................................................... 25 Figure 10: Validation of the SelS-V5 surrogate assay.................................................... 43 Figure 11: MS2 fragmentation of SelS-V5 Sec-containing peptide................................ 46 Figure 12: Efficiency of the SelS-V5 reporter................................................................ 50 Figure 13: Efficiency of the SelK-V5 reporter............................................................... 51 Figure 14: Position of the UGA-Sec codon affects UGA recoding in SelS-V5............. 54 Figure 15: The proximal region of the SelS 3’ UTR contains elements that affect ....................V5 expression............................................................................................... 57 Figure 16: The proximal SelS 3’ UTR contains two conserved regions......................... 58 Figure 17: The PSL and SPURdm do not affect mRNA levels or general ....................translation..................................................................................................... 61 v Figure 18: Deletion of the PSL and SPUR element mutation effects are not cell-type ..................... specific........................................................................................................ 63 Figure 19: Single point mutations in the SPUR element inhibit UGA recoding............ 66 Figure 20: Mutation of the SPUR element does not affect recoding directed by the .................... SelK or GPx4 SECIS elements..................................................................... 68 Figure 21: Deletion of the SelS PSL increases Sec insertion in the SelS-V5 .................... construct....................................................................................................... 71 Figure 22: Disruption of the SelS PSL by point mutation and loop deletions has no .................... effect on V5 expression................................................................................ 76 Figure 23: Other stem-loops can functionally replace the SelS PSL.............................. 77 Figure 24: A linear sequence can replace the SelS PSL.................................................. 78 Figure 25: Mutation of the SPUR element effects Sec insertion in FLAG-SelS ..................... but PSL deletion has no effect..................................................................... 81 Figure 26: Deletion of the PSL does not increase Sec insertion into the FLAG-SelS .................... construct....................................................................................................... 82 Figure 27: Relative position of SPUR element to UGA codon is important ....................f. ....................activity.......................................................................................................... 86 Figure 28: Mutations in the SECIS element rescue UGA recoding of the SPURdm..... 92 Figure 29: A protein binds to the SPUR element............................................................ 95 Figure 30: Model for the mechanism of action of the SPUR element..............................97 vi Acknowledgements It seems like not too long ago I was accepted into the Molecular Medicine program. At the beginning, I could not wait to start. There were times somewhere in the middle of grad school when it felt like time wasn’t moving at all. And now, at the end, I can’t tell you where all the time went and I cannot believe how fast it was over. Regardless of how long or short it felt, this was one of the best times of my life. I have met countless remarkable individuals, gained life-long friendships, and learned so much. Here I would like to thank the people who have helped me through this journey.
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