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Mechanism of Gene Regulation by Coding Polya Tracks Laura Lea Arthur Washington University in St

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Mechanism of Gene Regulation by Coding Polya Tracks Laura Lea Arthur Washington University in St Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2017 Mechanism of Gene Regulation by Coding PolyA Tracks Laura Lea Arthur Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biology Commons, Cell Biology Commons, and the Genetics Commons Recommended Citation Arthur, Laura Lea, "Mechanism of Gene Regulation by Coding PolyA Tracks" (2017). Arts & Sciences Electronic Theses and Dissertations. 1193. https://openscholarship.wustl.edu/art_sci_etds/1193 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Genetics and Genomics Dissertation Examination Committee: Sergej Djuranovic, Chair Nicholas Davidson Tim Schedl Jim Skeath Matthew Walter Mechanism of Gene Regulation by Coding PolyA Tracks by Laura Arthur A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2017 St. Louis, Missouri © 2017, Laura Arthur Table of Contents List of Figures ................................................................................................................................ iv List of Tables ................................................................................................................................. vi List of Abbreviations .................................................................................................................... vii Acknowledgments.......................................................................................................................... ix Abstract .......................................................................................................................................... xi Chapter 1: Introduction ................................................................................................................... 1 1.1 Poly(A) tail mediated regulation ...................................................................................... 2 1.2 Evidence for ribosomal stalling by polybasic sequences ...................................................... 6 1.3 Coding polyA sequences repress translation more than polybasic sequences ...................... 9 1.4 Perspectives ......................................................................................................................... 15 1.5 Figures ............................................................................................................................ 17 1.6 References ...................................................................................................................... 23 Chapter 2: Translational control by lysine-encoding A-rich sequences ....................................... 27 Preface ....................................................................................................................................... 27 2.1 Abstract .......................................................................................................................... 28 2.2 Introduction .................................................................................................................... 28 2.3 Results ............................................................................................................................ 30 2.4 Conclusion ...................................................................................................................... 38 2.4 Materials and Methods ................................................................................................... 39 2.4.1 Experimental Protocols ............................................................................................................. 39 2.4.2 Bioinformatic Analysis ............................................................................................................. 42 2.5 Figures ............................................................................................................................ 45 2.6 Tables ............................................................................................................................. 86 2.7 References .................................................................................................................... 133 Chapter 3: Rapid generation of hypomorphic mutations ............................................................ 135 Preface ..................................................................................................................................... 135 3.1 Abstract ........................................................................................................................ 136 3.2 Introduction .................................................................................................................. 136 3.3 Results .......................................................................................................................... 138 ii 3.4 Discussion .................................................................................................................... 152 3.5 Methods ........................................................................................................................ 156 3.6 Figures .......................................................................................................................... 168 3.7 References .................................................................................................................... 181 Chapter 4: Coding polyA tracks induce mRNA surveillance pathways ..................................... 186 4.1 Abstract ........................................................................................................................ 186 4.2 Introduction .................................................................................................................. 186 4.2 Results .......................................................................................................................... 188 4.3 Discussion .................................................................................................................... 193 4.4 Methods ........................................................................................................................ 194 4.5 Figures .......................................................................................................................... 197 4.6 References .................................................................................................................... 206 Chapter 5: Conclusions and Future Directions ........................................................................... 208 5.1 Summary ...................................................................................................................... 208 5.2 Future Directions .......................................................................................................... 208 iii List of Figures Figure 1.1 Normal and aberrant translation. ................................................................................. 18 Figure 1.2 Potential outcomes of programmed ribosome frameshifting on coding polyA tracks. 20 ....................................................................................................................................................... 21 Figure 1.3 Model of effects of synonymous mutations within coding polyA tracks .................... 22 Figure 2.1. Effects of different lysine codons on mCherry reporter expression and mRNA stability. ......................................................................................................................................... 46 Figure 2.2. The effect of codon usage in polylysine tracks on translation and protein levels. ..... 49 Figure 2.3. Native poly(A) tracks control reporter mRNA and protein levels. ............................ 52 Figure 2.4. The effect of synonymous mutations in poly(A) tracks of human genes. .................. 55 Figure 2.5. Putative mechanisms through which poly(A) tracks exert their function. ................. 58 Supplemental Figure 2.1. .............................................................................................................. 59 Supplemental Figure 2.2 ............................................................................................................... 60 Supplemental Figure 2.3 ............................................................................................................... 61 Supplemental Figure 2.4 ............................................................................................................... 63 Supplemental Figure 2.6. .............................................................................................................. 67 Supplemental Figure 2.7. .............................................................................................................. 69 Supplemental Figure 2.8. .............................................................................................................
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