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ABSTRACT Title of Dissertation: the IMPACT of TRANSLATIONAL ABSTRACT Title of Dissertation: THE IMPACT OF TRANSLATIONAL FIDELITY ON HUMAN HEALTH Carolina Marques dos Santos Vieira, Doctor of Philosophy, 2018 Dissertation directed by: Professor and Chair, Jonathan D. Dinman, Department of Cell Biology and Molecular Genetics Ribosomopathies are a class of diseases resulting from mutations in genes encoding ribosomal proteins and ribosome biogenesis factors. Pleiotropic clinical presentations of different ribosomopathies has been taken as evidence of specialized ribosomes. Alternatively, gene dosage effects have been proposed to account for the observed differences. A yeast genetics approach was used to address this issue. Due to a historical gene duplication event, S. cerevisiae cells harbor two ohnologs for most ribosomal proteins. Deletion of one yeast ribosomal protein ohnolog was used to mimic haploinsufficiency in diploid cells (i.e. pseudo-haploinsufficient yeast). Further, insertion of a second copy of the undeleted ohnolog into the locus of the deleted ohnolog enabled separation of effects due to gene dosage from those due to ribosomal protein ohnolog identity. We found that significant changes in translational fidelity in the ribosomal protein ohnolog deletion strains were corrected by ohnolog duplication. Changes in gene dosage, particularly as they may affect the abundance of an enzyme as central as the ribosome, can impart stress through far reaching effects on cellular metabolism. Thus, as an orthogonal approach, we also examined the stress profiles of cells harboring the cbf5-D95A allele (model of X-linked Dyskeratosis Congenita) and the rps23a-R69K allele (model of MacInnes Syndrome). RNA-seq analysis revealed increased expression of proteins involved in response to oxidative stress in cbf5-D95A cells. Growth curve analysis revealed a longer plateau of the cbf5-D95A cells upon reaching stationary phase, suggestive of a pre-adaptive stress response. Decreased ROS abundance, tunicamycin resistance and increased basal levels of HAC1 mRNA in the mutant cells support this hypothesis. Similar results were observed with regard to the ohnolog deletion strains. Taken as a whole, these data support the gene dosage model as opposed to the specialized ribosome hypothesis with the caveat that this conclusion is limited to yeast cells growing in rich medium. THE IMPACT OF TRANSLATIONAL FIDELITY ON HUMAN HEALTH by Carolina Marques dos Santos Vieira Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2018 Advisory Committee: Professor Jonathan D. Dinman, Chair Professor Najib El-Sayed Associate Professor Antony M. Jose Associate Professor Kan Cao Associate Professor Lisa Taneyhill, Dean’s Representative © Copyright by Carolina Marques dos Santos Vieira 2018 Dedication This dissertation is dedicated to my parents for all their love. They have been an immense source of inspiration and support in my life. Without them, I would not have made it this far. ii Acknowledgements I would like to thank my advisor, Dr. Jon Dinman, for the years of mentorship, support, patience, and encouragement. To my committee: thank you for your time and guidance. I would also like to thank past and present lab members of the Dinman lab, especially Dr. Joe Briggs, Dr. Denis Wafula, Dr. Ashton Trey Belew, Joe Kendra, Alex Olson, Swaksha Rachuri, Cassie Moomau, Dr. Alicia Cheek, Dr. Suna French, Dr. Sharmishtha Musalgaonkar, Dr. Vivek Advani, Dr. John Jones, Dr. Bin Chen. You have been great colleagues and friends. Thank you for the valuable discussions, experimental troubleshoot, and for the fun moments inside and outside of the lab. To all graduate students and post-docs I have interacted with during my time at the University: thank you both for your assistance with experiments and for your friendship. To all my friends who have helped me stay sane during my time in graduate school, thank you. A special thank you goes to Maria Osso and Lisa Starr for being some of my biggest cheerleaders. To the staff of the Department of Cell Biology and Molecular Genetics, especially to Moly Burke, Sharon Fabricante, Teresa Thompson, Dorothea O’Toole, Megan Leung, Priyanka Vengat, and Errica Philpott: thank you for all you have done for me, both from an administrative standpoint, but also as friends. iii I would like to thank my family for all the years of support, and for their patience as they watched me from an ocean away for the last nine years. Thank you for your understanding and unwavering love. I will forever be in your debt. Finally, a big thank you to my husband, Angel Galvez, for his love and support. Without him, none of this would have been possible. Thank you for helping me make it through to the end. Thank you, also, for always being there for me, for listening, and for making me laugh even when graduate school life got rough. This work was supported in part by NIH grant R01HL119439 to Dr. Jonathan D. Dinman, and three fellowships to Carolina Marques dos Santos Vieira (Graduate School Summer Research Fellowship, CMNS Dean’s Fellowship, MOCB-CA Summer Fellowship). Thank you to Dr. Susan Baserga and Dr. Sherif Abou Elela for kindly providing us with yeast strains, and to Dr. Alyson MacInnes for providing us with patient-derived cell lines. These were instrumental to completing the work discussed in this dissertation. iv Table of Contents Dedication ....................................................................................................... ii Acknowledgements ........................................................................................ iii Table of Contents ............................................................................................ v List of Tables ................................................................................................. vii List of Figures ................................................................................................ viii List of Abbreviations ........................................................................................ x Chapter 1: Introduction .................................................................................... 1 The ribosome ............................................................................................... 1 Ribosomopathies ......................................................................................... 3 Diamond Blackfan Anemia ....................................................................... 6 Isolated Congenital Asplenia .................................................................... 7 5q- Syndrome ........................................................................................... 8 X-linked Dyskeratosis Congenita .............................................................. 8 MacInnes Syndrome ................................................................................ 9 Ribosome biogenesis defects in ribosomopathies ....................................... 9 Ribosomopathies are associated with ribosome biogenesis defects ...... 12 Translational Fidelity .................................................................................. 15 Translational fidelity defects ....................................................................... 16 Programmed -1 Ribosomal Frameshifting (-1 PRF) ............................... 16 Programmed +1 Ribosomal Frameshifting (+1 PRF) ............................. 22 Missense and termination codon (nonsense) suppression ..................... 23 Translational fidelity reporters .................................................................... 23 Research overview .................................................................................... 25 Chapter 2: Materials and Methods ................................................................. 26 Preparation of plasmid DNA....................................................................... 26 Bacterial transformations ........................................................................... 26 NEB® 5-alpha competent E. coli ............................................................ 26 Stellar™ Competent Cells ...................................................................... 27 Yeast transformations ................................................................................ 27 Generation of UPF1 gene deletion yeast strains ........................................ 28 Generation of disruption cassette ........................................................... 28 Yeast transformation .............................................................................. 28 Verification of gene disruption by PCR ................................................... 29 CBF5 RNA-Seq experiments ..................................................................... 29 RNA extraction and cDNA sequencing ................................................... 29 Data quality assessment and visualization ............................................. 32 Differential expression and gene ontology analyses .............................. 33 Translational fidelity: Dual luciferase assays.............................................. 34 Yeast ...................................................................................................... 34 Mammalian cell lines .............................................................................. 35 Data analysis .........................................................................................
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