UC San Diego UC San Diego Electronic Theses and Dissertations Title Methods and Characterization of smORF-Encoded Microproteins Permalink https://escholarship.org/uc/item/25k5d8z0 Author Rathore, Annie Publication Date 2018 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA SAN DIEGO Methods and Characterization of smORF-Encoded Microproteins A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Annie Rathore Committee in charge: Professor Alan Saghatelian, Chair Professor Christopher Benner Professor Christopher Glass Professor Randolph Hampton Professor Martin Hetzer Professor Jens Lykke-Anderson 2018 © Annie Rathore, 2018 All rights reserved. The Dissertation of Annie Rathore is approved, and is acceptable in quality and form for publication on microfilm and electronically: Chair University of California San Diego 2018 iii TABLE OF CONTENTS Signature Page .......................................................................................................................... iii Table of Contents ....................................................................................................................... iv List of Figures ............................................................................................................................ vi Acknowledgements .................................................................................................................. viii Vita ............................................................................................................................................. x Abstract of the Dissertation ........................................................................................................ xi Chapter 1: Discovery and Emerging Biology of smORFs and Microproteins ............................... 1 1.1 Introduction ..................................................................................................................... 2 1.2 Functions of Small Open Reading Frames and Microproteins ......................................... 3 1.3 Discovery of Small Open Reading Frames and Microproteins ...................................... 12 1.4 Characteristics of Small Open Reading Frames and Microproteins ............................... 17 1.5 Approach for Functional Characterization ..................................................................... 19 1.6 Conclusion and Future Directions ................................................................................. 19 1.7 Figures ......................................................................................................................... 21 1.8 Acknowledgements ....................................................................................................... 28 1.9 References ................................................................................................................... 28 Chapter 2: Identification of Microprotein-Protein Interactions via APEX Tagging ....................... 40 Abstract ................................................................................................................................ 41 2.1 Introduction ................................................................................................................... 41 2.2 Results and Discussion ................................................................................................. 44 2.3 Conclusion .................................................................................................................... 49 2.4 Figures ......................................................................................................................... 50 2.5 Materials and Methods .................................................................................................. 67 2.6 Acknowledgements ....................................................................................................... 70 2.7 References ................................................................................................................... 71 Chapter 3: MIEF1 Microprotein Regulates Mitochondrial Translation ........................................ 75 Abstract ................................................................................................................................ 76 3.1 Introduction ................................................................................................................... 76 3.2 Results and Discussion ................................................................................................. 78 3.3 Conclusion .................................................................................................................... 86 iv 3.4 Figures ......................................................................................................................... 87 3.5 Materials and Methods .................................................................................................. 99 3.6 Acknowledgements ..................................................................................................... 106 3.7 References ................................................................................................................. 106 Chapter 4: Functional Characterization of StAR smORF ......................................................... 114 Abstract .............................................................................................................................. 115 3.1 Introduction ................................................................................................................. 115 3.2 Results and Discussion ............................................................................................... 117 3.3 Conclusion and Future Directions ............................................................................... 121 3.4 Figures ....................................................................................................................... 123 3.5 Materials and Methods ................................................................................................ 128 3.6 References ................................................................................................................. 131 v LIST OF FIGURES Chapter 1 Figure 1.1. uORF mediated regulation of downstream ORF expression .................................. 21 Figure 1.2. Biologically active microproteins ............................................................................ 23 Figure 1.3. Summary table for smORF-encoded biologically active microproteins .................. 24 Figure 1.4. Methods for smORF and microprotein discovery ................................................... 25 Figure 1.5. Web-Based integrated tools for smORF discovery and characterization ............... 26 Figure 1.6. Position of smORFs within RNAs .......................................................................... 27 Chapter 2 Figure 2.1. Schematic illustration of identification of microprotein- associated proteins by APEX tagging ................................................................................................... 50 Figure 2.2. Identification of MRI microprotein-protein interactions in live cells by APEX tagging .............................................................................................................. 51 Figure 2.3. Loading control of APEX and MRI-APEX FLAG immunoprecipitation ................ 52 Figure 2.4. Biotinylated proteomes of untransfected, APEX and MRI-APEX transfected cells .......................................................................................................................... 53 Figure 2.5. Loading control of MRI-APEX biotinylation ......................................................... 54 Figure 2.6. Identification of C11orf98 microprotein associated proteins .............................. 55 Figure 2.7. Schematic illustration of APEX control and C11orf98-APEX constructs ............ 56 Figure 2.8. Sub- cellular localization of biotinylated enriched proteins ................................. 57 Figure 2.9. Validation of C11orf98 microprotein interaction with NPM1 and NCL ................ 58 Figure 2.10. Loading control of C11orf98-APEX and APEX-C11orf98 biotinylation ............... 59 Figure 2.11. Validation of C11orf98 interaction with NPM1 and NCL by FLAG immunoprecipitation ........................................................................................... 60 Figure 2.12. Loading control of C11orf98-FLAG FLAG immunoprecipitation .......................... 61 vi Figure 2.13. Loading control of reciprocal anti-HA immunoprecipitation ................................ 62 Figure 2.14. Validation of C11orf98 interaction with NPM1 and NCL by reciprocal immunoprecipitation .......................................................................................... 63 Figure 2.15. Loading control of reciprocal anti-HA immunoprecipitation ................................ 64 Figure 2.16. C11orf98 microprotein was identified in public protein interaction database ....... 65 Figure 2.17. Co-localization of C11orf98 microprotein and NPM1 in cell nucleolus ................ 66 Chapter 3 Figure 3.1. MIEF1-MP expression and conservation ........................................................... 87 Figure 3.2. MIEF1-MP is a mitochondrial matrix microprotein ............................................