Functional Characterization of Selected Chloroplast RNA- Binding Proteins from Arabidopsis thaliana by Jan P. Kuciński A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular and Developmental Biology) in the University of Michigan 2020 Doctoral Committee: Associate Professor Andrzej T. Wierzbicki, Chair Professor Steven E. Clark Assistant Professor Peter L. Freddolino Professor Lyle A. Simmons Jan P. Kucinski [email protected] ORCID iD: 0000-0003-0550-5705 ©Jan P. Kucinski 2020 DEDICATION For my wife and children. ii ACKNOWLEDGEMENTS I wanted to express my gratitude to Andrzej for being my mentor over past six years. I have surely learned a lot and became a better scientist and possibly a better person. Thank you, Andrzej. To all Wierzbicki’s lab members. Specifically, Gudrun Boehmdorfer for being extremely helpful during my first years in the lab. She was not only a great teacher and mentor but also a supportive friend. Shriya Sethuraman and Hafiz Rothi for being fantastic friends over past five years. It was a blast working with you! Undergraduate students I had an honor to work with: Natalie Blackwood, Olivia Metcalf, Pragya Khurana and Matthew Dunn. Thank you for helping me with my projects and improving my teaching skills. To my Thesis Committee, I have enjoyed hearing your feedback and receiving your support and guidance throughout my candidacy. Thank you for the critical but supportive role you have played in my development as a scientist. To my dearest Family, you have always been my greatest support. If that was not for you I would not be anywhere close to where I am now. Specifically, to my Grandfather who have always believed in me and provided the best professional advices that I have ever gotten. iii TABLE OF CONTENTS DEDICATION ............................................................................................................................................... ii ACKNOWLEDGEMENTS ......................................................................................................................... iii LIST OF FIGURES .................................................................................................................................... vii ABSTRACT ................................................................................................................................................. ix CHAPTER I INTRODUCTION ........................................................................................................................................ 1 Plastids ..................................................................................................................................................... 1 Endosymbiotic origin of plastids ....................................................................................................... 1 Plastid development ........................................................................................................................... 2 Plastid division ..................................................................................................................................... 4 Plastid genome ....................................................................................................................................... 5 Plastid genome organization ............................................................................................................. 7 Plastid nucleoid ................................................................................................................................... 8 Plastid DNA replication ...................................................................................................................... 9 Plastid RNA metabolism ...................................................................................................................... 11 Organization of plastid-encoded genes ......................................................................................... 11 Transcription ...................................................................................................................................... 11 Transcription initiation ...................................................................................................................... 13 Intercistronic transcript processing ................................................................................................ 14 Splicing of plastid-encoded mRNAs .............................................................................................. 15 Transcription termination ................................................................................................................. 17 RNA processing .................................................................................................................................... 19 RNA editing ........................................................................................................................................ 19 RNA-binding proteins ........................................................................................................................... 20 PPR proteins ..................................................................................................................................... 20 RNA-stabilizing proteins .................................................................................................................. 22 RNA ends modifications and degradation ........................................................................................ 23 iv Endonucleolytic RNA processing ................................................................................................... 25 RNase H proteins ............................................................................................................................. 27 DeCL proteins ................................................................................................................................... 29 CHAPTER II RNase H1 is required for Arabidopsis thaliana embryonic development ............................... 31 Abstract .................................................................................................................................................. 31 Significance ........................................................................................................................................... 32 Introduction ............................................................................................................................................ 32 Results .................................................................................................................................................... 34 Origin of angiosperm RNases H1 .................................................................................................. 34 Origin of four RNases H1 in Arabidopsis ...................................................................................... 35 RNase H1-like proteins from Arabidopsis have canonical RNase H1 activity ........................ 36 RNH1A and RNH1B do not affect development .......................................................................... 37 Presence of RNH1B or RNH1C is required for viability .............................................................. 38 RNH1C is involved in chloroplast nucleic acid metabolism ....................................................... 39 Discussion .............................................................................................................................................. 41 Materials and Methods ......................................................................................................................... 44 Acknowledgements .............................................................................................................................. 50 Figures .................................................................................................................................................... 51 CHAPTER III Functional characterization of DeCL proteins from Arabidopsis thaliana .............................. 62 Abstract .................................................................................................................................................. 62 Significance ........................................................................................................................................... 63 Introduction ............................................................................................................................................ 63 Results .................................................................................................................................................... 68 DeCL proteins are present mostly in photosynthetic organisms ............................................... 68 Arabidopsis contains two plastid-localized DeCLs ...................................................................... 68 Plastid DeCLs are broadly expressed ........................................................................................... 70 DeCL1 and DeCL2 are required for plant survival ...................................................................... 70 DeCL1 interacts with plastid-encoded mRNAs ............................................................................ 71 Discussion .............................................................................................................................................