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Annie's Cute Little Helicase Growth-regulated expression and G0-specific turnover of the mRNA that encodes AH49, a mammalian protein highly related to the mRNA export protein UAP56 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anne M. Pryor, B.S. ***** The Ohio State University 2003 Dissertation Committee: Dr. Lee Johnson, Adviser Approved By Dr. Gustavo Leone Dr. David Bisaro Adviser Dr. Michael Ostrowski Ohio State Biochemistry Program ABSTRACT The expression patterns of many genes are regulated at the post- transcriptional level. Therefore, understanding the mechanisms of post- transcriptional processes such as splicing and nuclear export is extremely important. DExD/H box RNA helicases are involved in remodeling RNA-RNA and RNA-protein complexes in all aspects of mRNA metabolism. One DExD/H box protein, UAP56, is a presumed RNA helicase that has been shown to be essential for the nuclear export of all mRNAs in S. cerevisiae and D. melanogaster. I have identified another DExD/H box protein, AH49, which is 89.9% identical to UAP56. Although S. cerevisiae and D. melanogaster have only a single protein corresponding to this helicase (Sub2p and HEL, respectively), I show that mRNAs corresponding to AH49 and UAP56 are both expressed in human and mouse cells. Also, both proteins interact with the export factor Aly in GST pull down assays. UAP56 mRNA was more abundant than AH49 mRNA in many of the human and mouse tissues tested. However, AH49 mRNA was much more abundant than UAP56 mRNA in testes. UAP56 and AH49 mRNAs were present at similar levels in proliferating mouse 3T3 and human WI38 fibroblasts. However, when the cells were induced to enter quiescence, AH49 mRNA levels decreased 3-6-fold while UAP56 mRNA levels remained relatively constant. The amount of AH49 mRNA increased to the level ii found in proliferating cells within 5 hours after quiescent cells were stimulated to proliferate or following addition of the protein synthesis inhibitor, cycloheximide. AH49 mRNA was relatively unstable (T½ = 4 hr) in quiescent cells but was stabilized immediately following growth stimulation or addition of cycloheximide. In contrast, there was little change in the content or stability of UAP56 mRNA following growth stimulation. These observations show that two related RNA helicases, UAP56 and AH49, have differential gene regulation. The reason for this differential regulation may be because the two proteins have unique functions and remains to be determined. iii DEDICATION This work is dedicated to my wonderful husband whose help, love, and support enabled me to get through these difficult years. It is also dedicated to my family who never stopped praying for me and my experiments. Finally, it is dedicated to my adorable nieces and nephews who were a constant source of joy and entertainment. iv ACKNOWLEDGMENTS There are so many people who have given me invaluable help over the last 5 years that I can’t begin to thank them all. I would like to thank Dr. Johnson for being a wonderful, kind mentor. He has always had faith in me and supported me when I wanted to take my research in a new direction. I would also like to thank my other committee members for all of the time they spent brainstorming about my research. I would like to thank Fehmida Kapadia, David Armbruster, Alistar Harrison, Tom Rudge, Zhe Yang, and all the lab members who have given me so much help and advice. I would especially like to thank Mike Zianni whose real time PCR expertise made much of this work possible. I would like to thank Guo Wei and Maria Festing for their aid in retrieving mouse tissues, and Bethany Regner for her help with flow cyctometry. I especially want to thank Dr. Robin Reed (Harvard University) for sending me the GST-UAP56 and His-Aly expression plasmids, Dr. Michael Green (University of Massachusetts) for sending me the anti-GST-UAP56 antibodies, and Dr. Jas Lang (The Ohio State University) for giving me the human tissue RNA. v VITA June 09, 1975.........................................Born - Youngstown, Ohio 1998 .......................................................B.S. Biochemistry, Ohio University 1998-present ..........................................Graduate Teaching and Research Associate, The Ohio State University FIELDS OF STUDY Major Field: Ohio State Biochemistry Program vi TABLE OF CONTENTS ABSTRACT ...........................................................................................................ii DEDICATION .......................................................................................................iv ACKNOWLEDGMENTS ....................................................................................... v VITA .....................................................................................................................vi LIST OF FIGURES ...............................................................................................ix INTRODUCTION.................................................................................................. 1 Post-Transcriptional Processes and Regulation............................................... 2 Pre-mRNA Splicing .......................................................................................... 4 DExD/H box RNA helicases are involved RNA remodeling.............................. 6 Biological Role of UAP56 ............................................................................... 14 UAP56 is recruited during transcription...................................................... 15 UAP56 is recruited to the spliceosome. ..................................................... 17 UAP56 is involved in mRNA export............................................................ 18 Other Functions of UAP56 ......................................................................... 25 Introducing AH49 ........................................................................................... 25 Research Topic .............................................................................................. 32 MATERIALS AND METHODS............................................................................ 34 Plasmids......................................................................................................... 34 GST Pull Down Assay.................................................................................... 36 Mammalian Cell Culture ................................................................................. 38 RNA Isolation ................................................................................................. 39 Real Time PCR .............................................................................................. 39 Radioactive PCR............................................................................................ 47 Drug Studies .................................................................................................. 53 3H-Leucine Incorporation................................................................................ 54 3H-Uridine Incorporation................................................................................. 54 Flow Cytometry .............................................................................................. 55 RESULTS........................................................................................................... 56 UAP56 and AH49 interact with Aly................................................................. 56 vii Expression of UAP56 and AH49 mRNA in Tissues........................................ 62 UAP56 and AH49 mRNA Levels in Growth-Stimulated Cells......................... 74 Cycloheximide causes the UAP56 and AH49 mRNA levels to increase. ....... 86 Half-lives of UAP56 and AH49 mRNA............................................................ 93 Additional Drug Studies................................................................................ 100 Flow Cytometry ............................................................................................ 104 Alternatively Spliced Forms.......................................................................... 108 Yeast Complementation Assay .................................................................... 111 DISCUSSION ................................................................................................... 113 UAP56 and AH49 Gene Regulation ............................................................. 115 AH49 mRNA Turnover ................................................................................. 124 AH49 may be important in the testes. .......................................................... 132 UAP56 and AH49 Protein Levels ................................................................. 133 Yeast Complementation Assay .................................................................... 134 RNA Interference ......................................................................................... 139 Protein-Protein and Protein-RNA Interactions.............................................. 140 Alternatively Spliced Forms of UAP56 and AH49......................................... 141 An Unknown Protein 50% Identical to UAP56.............................................. 142 Conclusion ................................................................................................... 146 PREQUEL: Elucidating the role of the transcription factor LSF in thymidylate synthase gene regulation. ...............................................................................
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