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Copyright by Sanjeev Vinayak Namjoshi 2017 The Dissertation Committee for Sanjeev Vinayak Namjoshi Certifies that this is the approved version of the following dissertation: Bioinformatic Approaches to Screening the Molecular Framework Underlying Local Dendritic mRNA Translation Committee: Johann Hofmann Supervisor Kimberly F. Raab-Graham Co- Supervisor Nigel S. Atkinson Edward M. Marcotte Christopher S. Sullivan Bioinformatic Approaches to Screening the Molecular Framework Underlying Local Dendritic mRNA Translation by Sanjeev Vinayak Namjoshi, B.S.Bio Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2017 Dedication I dedicate this work to all those who have been patient and supportive of me for over half a decade. To Dr. Kimberly Raab-Graham for her guidance and vision which has nurtured me into the scientist I am today. To the Raab-Graham lab for listening to my rambling discourse, bad music, and being a clarifying force in moments of confusion. To my mother for her unending supply of wisdom and council so I could better understand myself. To my father for questioning my own assumptions so that I could become a better scientist. To my sister for helping me realize what it is I believe. To my friends: Ariana, Alex & Alex, Jennifer, Kalen, Leslie, Peter, and Rick, who always inquired about my progress and emboldened me to move forward. To Elissa Jo for her constant encouragement and unwavering optimism at the most difficult moments. Finally, I dedicate the following quote to all the rodents whose heads have been sacrificed for my projects: “The world to which we have belonged offers nothing to love outside of each individual insufficiency: its existence is limited to utility […] Existence is not only an agitated void, it is a dance that forces one to dance with fanaticism. Thought that does not have a dead fragment as its object has the inner existence of flames […] Human life is exhausted from serving as the head of, or the reason for, the universe. To the extent that it becomes this head and this reason, to the extent that it becomes necessary to the universe, it accepts servitude. If it is not free, existence becomes empty or neutral and, if it is free, it is in play. The Earth, as long as it only gave rise to cataclysms, trees, and birds, was a free universe; the fascination of freedom was tarnished when the Earth produced a being who demanded necessity as a law above the universe. Man, however, has remained free not to respond to any necessity; he is free to resemble everything that is not himself in the universe. He can set aside the thought that it is he or God who keeps the rest of things from being absurd. Man has escaped from his head just as the condemned man has escaped from his prison […] Beyond what I am, I meet a being who makes me laugh because he is headless […] The fate and the infinite tumult of human life is open not to those who exist like gouged out eyes, but to those who are like seers, carried away by a shattering dream that could not belong to them.” -Georges Bataille, La Conjuration Sacree (1937) Acknowledgements I would like to acknowledge members of the Raab-Graham lab, past and present, who have helped me along the way and contributed to my continued success and dedication: Chelcie Heaney, Emily Workman, Farr Niere, Luisa Cacheaux, Natasha Sosanya, Sarah Wolfe, and all rotation students and undergrads that have passed through the lab the last 3 and a half years. Thanks to Dr. Hans Hofmann his mentorship and advice. Thanks to my committee for their guidance and helpful discussion. And finally, thanks to Dr. Kimberly Raab- Graham for taking a chance on me. Thank you for all you have done. v Bioinformatics Approaches to Screening the Molecular Framework Underlying Local Dendritic mRNA Translation Sanjeev Vinayak Namjoshi, Ph.D The University of Texas at Austin, 2017 Supervisor: Johann Hofmann Co-Supervisor: Kimberly F. Raab-Graham Learning and memory systems within the brain are believed to be the result of molecular events that occur at the synapse. These molecular events control synaptic efficacy – the modulation of the underlying molecular architecture of the synapse to influence the strength or weakness of connections between pre- and postsynaptic neurons. Deficits in any of these molecular processes results in neuronal dysfunction. The Mammalian/Mechanistic Target of Rapamycin Complex (mTORC1) is a protein complex within the brain composed of the serine/threonine kinase mTOR and other interacting proteins. Dysregulation of mTOR disrupts the many processes under its control such as local dendritic translation at the synapse. Since mTOR is at the core of many important signaling pathways, aberrant mTOR activity results in neuronal disease. The network of interactions between these molecular components is vast, forming an interconnected system that is dynamic and directed. In order to better understand the mechanistic nature of these interactions, the application of high-throughput technologies must be employed. Here we utilize multiple bioinformatics approaches combined with high-throughput technologies to clarify the role of mTOR in local dendritic translation. Using mass spectrometry, we provide the first evidence that mTOR bidirectionally controls the vi expression of over 700 proteins in the cortex, many of which are known to be associated with diseases in which mTOR is overactive (Chapter 2). Our investigations reveal a novel role PARK7 in tuberous sclerosis complex. Furthermore, we use and develop multiple bioinformatics tools to further delineate the nature of the RNA-binding properties of PARK7 and alternative avenues for drug discovery (Chapter 3). Finally, we provide evidence that the Fragile-X Mental Retardation protein sequesters a population of mRNAs involved in trans-synaptic signaling that mTOR translates to remodel the synapse, providing a mechanistic basis for the action of rapidly-acting antidepressants (Chapter 4). Collectively, our work stresses the importance of applying high-throughput technologies to answer long-standing questions in the field of local dendritic translation. Our findings provide new avenues of investigation and research to better understand neuronal disease and synaptic plasticity. vii Table of Contents List of Tables ......................................................................................................... xi List of Figures ....................................................................................................... xii Chapter 1: Introduction ............................................................................................1 Local Translation and Dendritic RNA Transport ...........................................1 mTOR and the Control of Local Translation ..................................................3 Using high-throughput assays to understand the molecular basis of memory9 Experimental design of high-throughput assays ...........................................10 Rationale for extracting cell-specific and subcellular RNA populations......12 What isolating regulatory factors can tell you about synaptic efficacy ........17 High-Throughput Assays ..............................................................................28 Combined transcriptomics and proteomics ...................................................41 From statistical models to scientific models: experimental validation and visualization of identified proteins and RNA ......................................50 How high throughput assays may answer long-standing questions in the field of learning and memory by identifying novel proteins involved in synaptic plasticity ...............................................................................................64 Motivation for studies ...................................................................................66 Chapter 2: Rapid repression of mTORC1 shifts the regional expression of disease- related protein ensembles, .............................................................................70 Introduction ...................................................................................................70 Experimental Procedures ..............................................................................72 Results ...........................................................................................................81 Discussion .....................................................................................................95 Supplemental Figures..................................................................................100 Supplemental Tables ...................................................................................102 Chapter 3: Bioinformatic approaches identify PARK7 as a novel protein aberrantly expressed in Tuberous Sclerosis Complex, .................................................104 Introduction .................................................................................................104 Experimental Procedures ............................................................................106 viii Results .........................................................................................................111 Discussion ...................................................................................................124 Supplemental Tables ...................................................................................126