Analysis of the Pumilio Repression Mechanism and its Impact on the Transcriptome A Dissertation SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Rebecca J. Haugen IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor Dr. Aaron Goldstrohm Committee members: Dr. Anja Bielinsky, Dr. Eric A. Hendrickson, Dr. David Greenstein May 2021 Rebecca J. Haugen Copyright 2021 Acknowledgements I would like to begin by acknowledging my husband Travis, for his ceaseless and unwavering support on this long journey. None of this would have been possible without him. I also thank my kids, Cody, Sarah, and Grace, for sacrificing some of their “Mom time” to all-day writing sprees or late nights in the lab. To Cody, for showing a keen interest in the science and allowing me to practice explaining it to him; to Sarah, for being my angel of sanity with relentless encouragement; to Grace, for providing comedic relief, and also engaging in late-night science talks: you have all been more instrumental in this accomplishment than I could ever explain. I would also like to thank my parents for their support and encouragement. I would like to thank my advisor Dr. Aaron Goldstrohm, for his guidance, and seemingly bottomless reservoir of patience and flexibility. I also thank my committee members Drs. Anja Bielinsky, Eric A. Hendrickson, and David Greenstein, for their encouragement, help, and feedback. I would like to especially thank Dr. Greenstein for supporting me and believing in me. I want to also thank the members of the Goldstrohm lab, past and present for their insight, encouragement, and scientific discussion, especially Dr. Katie McKenney, and my bench-mate, Rob Connacher. i Dedication This work is dedicated to my daughters, Sarah and Grace, and my son Cody. May you find enjoyment in working hard for something. ii Abstract The expression of genes in an organism is controlled by many methods. One of the most important means of regulation is post-transcriptional control of messenger RNA (mRNA). RNA binding proteins (RBPs) are crucial for this process. Here we describe the characterization of a functional domain within the Drosophila RBP Pumilio (Pum) and examine the effects of Pum on the transcriptome. Pum proteins are a highly conserved family of RBPs that control the expression of mRNA by binding to specific motifs in the 3’-UTR of transcripts. Through associations with other proteins, Pum proteins regulate the fate of an RNA, primarily causing decay of the target. Drosophila Pum contains autonomous domains capable of enacting decay independently from its RNA binding domain. The most conserved domain is repression domain 3 (RD3) which we characterize here. Using conservation analysis, we define conserved regions within RD3, and show their importance for the repressive activity of RD3. We identify two specific amino acids, F1033 and F1040, which are necessary for RD3’s function. Using yeast two-hybrid assays, we show that RD3 contacts several members of the CNOT deadenylase complex, the central scaffold unit Not1, and subunits Not2 and Not3. In Drosophila cells, we show that RD3 can interact with the CNOT complex. We further explore the endogenous targets of Drosophila Pum proteins by performing RNA sequencing experiments in which we deplete Pum and members of the CNOT complex. This is the first global analysis of the functional regulation of Pum targets in Drosophila. From this analysis we find that thousands of genes rely upon the regulatory framework provided by Pum. We demonstrate specific regulation of several targets of Pum using reporter assays and identify functional Pum binding sites within target 3’-UTRs. Finally, we show that regulation by Pum also affects the levels of expressed protein for two important targets, Pde11 and Raf. The work shown here enhances our understanding of Pum proteins for all organisms and the field of post- transcriptional control. iii Table of Contents Acknowledgements ............................................................................................... i Dedication .............................................................................................................ii Abstract ................................................................................................................ iii Table of Contents .................................................................................................iv List of Tables ....................................................................................................... vii List of Figures ..................................................................................................... viii List of Abbreviations ............................................................................................ xii Chapter 1: Introduction ......................................................................................... 1 1.1. Making the mRNA ................................................................................................ 1 1.2. Characteristics of mRNA ...................................................................................... 5 1.3. Cytoplasmic fates of mRNA ................................................................................. 8 1.3.1. Translation ..................................................................................................... 8 1.3.2. Storage and localization ................................................................................ 9 1.3.3. Messenger RNA decay .................................................................................10 1.4. Pumilio and PUF proteins....................................................................................13 1.4.1. Essential roles of Pum proteins ....................................................................14 1.4.2. Regulation of Pum ........................................................................................17 1.4.2.1. Regulation of Pum RNA .........................................................................17 1.4.2.2. Regulation of Pum protein ......................................................................18 1.4.3. Protein interactors of Pum ............................................................................20 1.4.4. The structure of Pum proteins .......................................................................21 Chapter 2: Structure-function analysis of RD3.................................................... 24 2.1. Introduction .........................................................................................................24 2.2. Overall structure of RD3 ......................................................................................26 2.3. Identification of conserved residues in RD3 ........................................................26 2.4. Functional activity of conserved regions ..............................................................31 2.4.1. Truncations of RD3 .......................................................................................32 2.4.2. Deletions of conserved regions .....................................................................36 2.4.3. Alanine scanning of conserved regions ........................................................40 2.4.4. Sufficiency of CR1 and CR4 .........................................................................46 2.5. Summary and discussion ....................................................................................49 2.6. Materials and methods ........................................................................................52 Chapter 3: Interactions of RD3 with the CNOT complex .................................... 56 iv 3.1. Introduction .........................................................................................................56 3.2. Interaction screens - Y2H assays ........................................................................57 3.3. Immunoprecipitation assays in Drosophila cells ..................................................71 3.4. Summary and discussion ....................................................................................76 3.5. Materials and methods ........................................................................................78 3.5.1. CRISPR generation of Not1-Flag Dl1 cells ...................................................82 Chapter 4: Endogenous targets of Pum proteins ................................................ 86 4.1. Introduction .........................................................................................................86 4.2. Identification of endogenous Pum targets ...........................................................87 4.3. Validation of Pum targets ....................................................................................91 4.4. Contribution of RD3 to repression of endogenous targets ...................................92 4.5. Pum depletion causes an increase in target protein levels ..................................97 4.6. Not1 as the effector of Pum-mediated decay of endogenous targets ..................99 4.7. Summary and discussion .................................................................................. 101 4.7.1. Biological significance of targets ................................................................. 102 4.8. Materials and methods ...................................................................................... 106 4.8.1. Quantitative western blotting ......................................................................