Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2017 Regulation of gene expression by RNA binding proteins and microRNAs Kyle Cottrell Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biochemistry Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Cottrell, Kyle, "Regulation of gene expression by RNA binding proteins and microRNAs" (2017). Arts & Sciences Electronic Theses and Dissertations. 1192. https://openscholarship.wustl.edu/art_sci_etds/1192 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 Cell Biology Dissertation Examination Committee: Sergej Djuranovic, Chair Kathleen Hall Tim Schedl Sheila Stewart Andrew Yoo Regulation of gene expression by RNA binding proteins and microRNAs by Kyle Aaron Cottrell 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, Kyle Aaron Cottrell Table of Contents List of Figures .............................................................................................................................. vii List of Tables ................................................................................................................................ ix Acknowledgments ......................................................................................................................... x Abstract ........................................................................................................................................ xii Chapter 1: Introduction ............................................................................................................... 1 1.1 The messenger RNA ............................................................................................................. 1 1.2 Translation ............................................................................................................................. 3 1.3 Decay ..................................................................................................................................... 5 1.4 Post-transcriptional Regulation ............................................................................................. 5 1.4.1 microRNAs .................................................................................................................................. 5 1.4.2 RNA binding proteins .................................................................................................................. 8 1.5 Conclusions ......................................................................................................................... 11 1.6 References ........................................................................................................................... 13 Chapter 2: Urb-RIP – an adaptable and efficient approach for immunoprecipitation of RNAs and associated RNAs/proteins ........................................................................................ 22 Preface ....................................................................................................................................... 22 2.1 Abstract ............................................................................................................................... 23 2.2 Introduction ......................................................................................................................... 23 2.3 Materials and Methods ........................................................................................................ 26 2.3.1 Construction of 2HA-Urb and SLII-tagged RNA constructs ..................................................... 26 2.3.2 Cell culture and transfection ...................................................................................................... 27 2.3.4 Crosslinking and immunoprecipitation of SLII-tagged RNA (Urb-RIP) ................................... 28 2.3.5 RNA analysis by RT-qPCR ....................................................................................................... 30 2.3.6 Protein analysis by western blotting .......................................................................................... 30 2.4 Results ................................................................................................................................. 31 2.4.1 General description of Urb-RIP ................................................................................................. 31 2.4.2 Urb-RIP allows for enrichment of mRNAs ............................................................................... 33 2.4.3 Urb-RIP enriches for tagged mRNA and a bound RNA binding protein .................................. 35 iii 2.4.4 Urb-RIP enriches for miRNAs and Ago2 .................................................................................. 36 2.4.5 Urb-RIP confirms binding of PABP to the RNAPIII lncRNA BC200 ...................................... 37 2.5 Discussion ........................................................................................................................... 39 2.6 References ........................................................................................................................... 43 2.6 Supplemental Information ................................................................................................... 49 2.6.1 Detailed Protocol........................................................................................................................ 49 2.6.2 Supplementary Figures .............................................................................................................. 52 2.6.2 Supplementary Tables ................................................................................................................ 58 Chapter 3: Translation efficiency is a determinant of the magnitude of miRNA-mediated repression ..................................................................................................................................... 63 Preface ....................................................................................................................................... 63 3.1 Abstract ............................................................................................................................... 64 3.2 Introduction ......................................................................................................................... 65 3.3 Results ................................................................................................................................. 66 3.3.1 The 3’UTR modulates translatability and miRNA-mediated repression ................................... 66 3.3.1 miRNA-mediated repression is modulated by changes in codon optimality and 5’UTR .......... 69 3.3.2 Translation efficiency is a determinant of miRNA-mediated repression ................................... 72 3.4 Discussion ........................................................................................................................... 75 3.5 Materials and Methods ........................................................................................................ 80 3.5.1 Construction of Reporters .......................................................................................................... 80 3.5.2 Transfection and Luciferase Assay ............................................................................................ 82 3.5.3 RNA Analysis ............................................................................................................................ 83 3.5.4 Cell Culture ................................................................................................................................ 84 3.5.5 Analysis of Ribosome Profiling and RNA-seq .......................................................................... 84 3.5.6 Model fitting .............................................................................................................................. 85 3.6 References ........................................................................................................................... 86 3.7 Supplemental Information ................................................................................................... 94 3.7.1 Renilla Luciferase Coding Sequences ........................................................................................ 94 3.7.2 Supplemental Figures ................................................................................................................. 97 3.7.3 Supplemental Tables ................................................................................................................ 115 Chapter 4: PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs 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