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Trna Subcellular Dynamics Dictates Modification and Nutrient Sensing

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Trna Subcellular Dynamics Dictates Modification and Nutrient Sensing tRNA subcellular dynamics dictates modification and nutrient sensing DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Alan Christopher Kessler Graduate Program in Microbiology The Ohio State University 2018 Dissertation Committee: Dr. Juan D. Alfonzo, Advisor Dr. Jane E. Jackman Dr. Charles Daniels Dr. Patrice Hamel Copyright by Alan C. Kessler 2018 Abstract In all eukaryotes, tRNAs are transcribed in the nucleus and then exported to the cytoplasm to engage in protein synthesis. However, previous work in Saccharomyces cerevisiae showed that tRNAs can also be sent back to the nucleus, and intracellular transport can be altered in response to starvation, leading to nuclear accumulation of tRNA. At least in one case, retrograde nuclear transport from the cytoplasm is necessary for wybutosine formation in tRNAPhe. Despite the fact that retrograde transport has been firmly established in yeast, it has been difficult to assess whether such a mechanism has a broader evolutionary distribution in eukaryotes. In the first part of this dissertation, I examined the post-transcriptional modification Queuosine (Q), and its relationship to retrograde transport in Trypanosoma brucei. Q is found at the first position of the anticodon in several tRNAs (tRNATyr, tRNAAsp, tRNAAsn and tRNAHis) and is presumably important for protein synthesis, although its function is not yet fully understood. Eukaryotes cannot synthesize Q and must rely on uptake and salvage of the free base from either nutrients or from gut microbiota. Following uptake, the enzyme tRNA guanine-transglycosylase (TGT) is responsible for the incorporation of queuine into tRNA by replacing guanine. In this work I show that T. brucei’s TGT (TbTGT) is a nuclear enzyme essential for Q formation in tRNA. One of its natural substrates, tRNATyr, also contains an intron, which must be removed prior to Q formation. In the present work, I show that because essential components of the ii splicing machinery are cytoplasmic, there is a dynamic interplay between tRNA splicing and modifications, all driven by the intracellular distribution of the different maturation components. Taken together, I demonstrate the existence of a tRNA nuclear retrograde transport pathway in T. brucei akin to what has been described in yeast, but with implications for other eukaryotic systems. The latter half of the dissertation examines Q and its potential involvement in nutrient sensing in T. brucei. As T. brucei transitions from the procyclic insect stage, to the mammalian bloodstream stage, metabolic reprograming occurs, with concomitant changes in the expression of stage specific genes. Additionally, in T. brucei gene regulation occurs post-transcriptionally. Because of this, post- transcriptional modifications may play critical roles in regulating gene expression. Here I show that Q modification levels change between the procyclic and bloodstream developmental stages of the parasite. Q levels also fluctuate in response to changes in the availability of a subset of amino acids. These findings have implications for how organisms may use modifications to sense nutrient availability and adjust translational rates accordingly. iii Dedication I would like to dedicate this work to my loving mother Judy Kessler. Without her guidance and unwavering support, none of this would have been possible. iv Acknowledgments It would be impossible to acknowledge everyone who had a positive impact on my professional development during graduate school. First off, I would like to thank Dr. Juan Alfonzo and Dr. Mary Anne Rubio. They have both created an environment that has been challenging yet rewarding in all its aspects. From the interesting RNA topics, to the numerous political conversations we have had, it has all been intellectual and enjoyable. I appreciate all the jokes over the years which made failed experiments and mistakes seem light hearted and easier to deal with. The lab could not be described without the different friends that I have grown accustomed to over the years. One of my best friends in the lab, Katie McKenney, has been there for me throughout all the ups and downs experienced throughout graduate school. I appreciate all the moments she helped me with experiments, as well as her unwavering positive outlook. I would next like to thank Dr. Ian Fleming for his guidance and friendship. I will never be able to “pay the bills” as he once put it, but I am thankful for all his assistance in the lab and for “voting to allow me to say on that side of the lab.” There were also many others in the lab over the years which I have fond memories with including Dr. Zdeněk Paris, Dr. Paul Sample, Dr. Raphael Sores, Scott Hinger, Caitlin Moore, Gabriel d’Almeida. None of this would have been possible without the love and support that my family and friends have given me over the years. My mother Judy Kessler, has always encouraged me to find the right path in my life. She has been full of advice v and above all, helped me succeed in nearly every way imaginable. I was fortunate to have Michelle Gibbs always around through most of my graduate schooling. She has been caring, thoughtful, and made the experience more enjoyable. I would also like to mention my late father Christopher Kessler who has been a major source of inspiration throughout my life and late grandmother Adeline Kessler, who always encouraged me to never stop learning. vi Vita 2008 ....................................................... Kane Area High School 2012 ....................................................... B.S. Biology, Indiana University of PA 2012 to present ....................................... Graduate Teaching/Research Associate, Department of Microbiology, The Ohio State University Publications Kessler AC, Kulkarni SS, Paulines MJ, Rubio MAT, Limbach PA, Paris Z, Alfonzo JD. Retrograde nuclear transport from the cytoplasm is required for tRNATyr maturation in T. brucei. RNA Biology. 2017. Sep 13:1-9. ahead of print. Kessler AC, d’Almeida GS, Alfonzo JD. The role of intracellular compartmentalization on tRNA processing and modification. RNA Biology. 2017. Aug 29:1-13. ahead of print. Lopes RR, Silveria Gde O, Eitler R, Vidal RS, Kessler A, Hinger S, Paris Z, Alfonzo JD, Polycarpo C. The essential function of the trypanosoma brucei Trl1 homolog in procyclic cells is maturation of the intron-containing tRNATyr. RNA. 2016. 22(8):1190-9. Lopes RR, Kessler AC, Polycarpo C, Alfonzo JD. Cutting, dicing, healing and sealing: The molecular surgery of tRNA. Wiley Interdiscip. Rev. RNA. 2015. 6(3):337-49. Fields of Study Major Field: Microbiology vii Table of Contents Abstract ............................................................................................................. ii Acknowledgments ............................................................................................. v Vita ................................................................................................................... vii Publications ...................................................................................................... vii Fields of Study ................................................................................................. vii Table of Contents ............................................................................................ viii List of Tables .................................................................................................... xii List of Figures ................................................................................................... xii Chapter 1 : Introduction ..................................................................................... 1 1.1 A brief Introduction to Trypanosoma brucei ................................................. 1 1.2 Gene regulation in T. brucei ........................................................................ 4 1.3 Transfer RNA .............................................................................................. 6 1.4 Role of compartmentalization on tRNA processing and modification ........ 10 1.4.1 Functionality checkpoints: Early steps in tRNA maturation in eukaryotes ..................................................................................................................... 16 1.4.2 The cytosolic fate of tRNA modifications ............................................. 22 1.4.3 The connection between tRNA splicing and modifications .................. 25 viii 1.4.4 Intracellular transport dynamics that set the order of modifications .... 31 1.4.5 Modifications and the Mitochondria ..................................................... 39 1.5 The Queuosine Modification ...................................................................... 46 1.5.1 The Queuosine Pathways of Bacteria and Eukarya ............................ 47 1.5.2 TGT in Bacteria and Eukarya .............................................................. 50 1.5.3 The Biological Role of Q ..................................................................... 52 Chapter 2 : Retrograde transport is required for tRNA maturation in T. brucei ........................................................................................ 56 2.1 Introduction ............................................................................................... 57 2.2 Results ...................................................................................................... 60 2.2.1
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