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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9* black and white photographic prints are available for any photographs or Uustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road, Atm Arbor, Ml 48106-1346 USA 800-521-0600 UMT MOLECULAR ANALYSIS OF L7/PCP-2 MESSENGER RNA AND ITS INTERACTING PROTEINS DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in Ohio State Biochemistry Program of The Ohio State University By Xulun Zhang, M.S. ***** The Ohio State University 2001 Dissertation Committee: Approved by Professor John D. Oberdick, Adviser Professor Kathleen Boris-Lawrie - Professor Michael Ostrowski Adviser Professor Harald Vaessin Ohio State Biochemistry Program UMI Number 3031292 UMI" UMI Micrafbrm 3031292 Copyright 2002 by Bell & HoweM Information and Learning Company. All rights reserved. This microfbrm edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Laammg Company 300 North Zeeb Road P.O. Box 1346 Arm Arbor. Mi 48ia6-1346 ABSTRACT L7 protein is a G protein a subunit modulator that is specifically expressed at high levels in cerebellar Purkinje cells. L7 mRNA has been shown to be localized in both cell bodies and dendrites in Purkinje cells, but the mechanisms of L7 mRNA localization, translation and the regulation of its translation in dendrites remain unclear. Here we present data to show that L7 mRNA has two different transcripts due to the choice of two alternative first exons, and this property is A conserved in rodent and in human. Both transcript forms are found in both cell bodies and dendrites in human and in rodent, and the distribution of the two transcripts in Purkinje cells is developmentally regulated. These two transcripts in turn will give rise to two L7 protein isofdnns. The shorter form contains one GoLoco motif, a motif which has been shown to interact with GDP-bound Go, whereas the longer form has two GoLoco motik. A ds-element which is responsible for the formation of an RNA-protein complex has been identified in the L7 3' UTR, and this element is conserved in rodent and in human. Two of the protein components of the RNA-protein complex have been partially purified by FPLC and they have been identified as CGI-145 and UNR protein. CGI-145 has no known function so far, but UNR has been shown elsewhere to be involved in cap-independent translational control and/or mRNA stability. Dedicated to my family III ACKNOWLEDGMENTS I wish to thank my adviser, Dr. John Oberdick , for intellectual support, encouragement, and enthusiasm which made this thesis possible, and for his patience in correcting both my stylistic and scientific errors. I would like to thank my dissertation committee members. Dr. Michael Ostrowski, Dr. Harald Vaessin and Dr. Kathleen Boris-Lawrie for their support and inspiring discussions. I would like to thank the Ohio State Biochemistry Program and the Ohio State University for their support. I would like to thank all of my colleagues Dr. Stephan Baader, Dr. Feng Bian, Dr. Teresa Chu, Yufang Zhang and Hailing Zhang for their help in the experiments and inspiring discussions. I would like to thank my wife Haidong and my daughter Yaning for their support during the whole work. IV VITA March 11,1966 ..................................................................Bom in Tianjin, P.R.China 1984 - 1988 .................................................... B.S. Biochemistry, NanKai University 1988 -1 9 9 1 ....................................................M.S. Biochemistry, Nankai University 1991 -1 9 9 5 ....................................................Research Assistant, Chinese Academy of Medical Sciences 1995 - present .............................................. Graduate Research Associate, The Ohio State University PUBUCATIONS 1 ) Zhang,X.; Baader,S.L; Bian,F.; Muller,W.; Oberdick,J. High level Purkinje cell specific expression of green fluorescent protein in transgenic mice Histochem.Cell Biol. 115,455-464,2001 2) Baader, S. L, Vogel, M. W., Sanlioglu, S., Zhang, X., Oberdick, J., Selective disruption of "late onset* sagittal banding patterns by ectopic expression of Engrailed-2 in cerebellar Purkinje cells. J. Neurosci. 19, 5370- 5379,1999 3) Sanlioglu, S., Zhang, X., Baader, S. L, Oberdick, J., Regulation of a Purkinje cell-specific promoter by homeodomain proteins: repression by Engrailed-2 vs. synergistic activation by HoxaS and Hoxb7. J. Neurobiol. 3 6 ,559- 571, 1998 4) Zhang, X., Wang, Y., Streptomyces mycarofaciens midecamycin 4*-0- propionyltransferase gene structure. Weishengwu Xuebao 36,417-422,1996 5) Gao, C., Zhang, X., Cai, B., Li, X., Qiao, M., Zhang, X., Copy number determination and restriction map of plasmid pNK866. Weishengwu Xuebao 34, 81-84, 1994 6) Yang, Y., Zhang, X., Yu, Y., Guo, X., Study on immobilization of DNA on VT copolymer. Lizi Jiaohuan Yu Xifu 6,172-177,1990 FIELDS OF STUDY Major Field: Biochemistry VI TABLE OF CONTENTS Abstract ........................................................................................................................ii Dedication .................................................................................................................... iii Acknowledgments ...................................................................................................... iv Vita ............................................................................................................................... V List of Tables ............................................................................................................ viii List of Figures ............................................................................................................. ix Chapters: 1. Introduction............................................................................................................1 1.1 Cerebellum serves as an Ideal model for the study of the nervous system ..............................................................................................1 1.2 L7/pcp-2 is specifically expressed in cerebellar Purkinje cells ................... 4 1.2.1 Discovery of L7 gene ..............................................................................4 1.2.2 L7 expression in cerebellum is regulated spatially and temporally, and L7 promoter is a valuable cell type specific expression tool 5 1.3 The expression of L7 is regulated by thyroid hormone and other factors..9 1.3.1 RORa regulates L7 expression .............................................................. 9 1.3.2 Evidence of other L7 gene regulators ................................................. 10 VII 1.4 L7 protein interacts with Goi and Goo modulating GDP release from Go...................................................................................................... 11 1.4.1 L7 knockout animals showed no significant phenotype ................... 11 1.4.2 L7 protein interacts with Go subunit .................................................. 12 1.5 L7 protein contains GoLoco motif ..............................................................14 1.6 RGS proteins belong to a protein kwnily and undertake multiple functions including GAP and GDI .............................................................................. 15 1.6.1 RGS proteins are stemmed from yeast Sst2 protein and are present in other lower eukaryotic organisms .................................................. 16 1.6.2 Mammalian RGS proteins are grouped into subfamilies ..................17 1.7 AGS proteins .............................................................................................. 23 1.8 Other GoLoco motif-containing proteins ...................................................24 1.9 GoLoco motif is the interaction site for Gasubunit and it acts as GDI 25 1.10 L7 mRNA is localized in both cell body and dendrites in Purkinje cells..28 1.11 Cis-elements and trans-factors mediate mRNA translocation ............... 30 1.11.1 Cis-eiement is required for mRNA translocation in neurons ......... 31 1.11.2 Protein factors are involved in RNAIocalization in dendrites ......... 32 1.12 Local protein synthesis at synapse in dendrites ....................................33 1.13 Work presented here ............................................................................... 36 2. Material and methods .....................................................................................