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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 ...... 41
viii 2.1 Mouse strains ...... 41
2.2 Rat strains ...... 41
2.3 Human Cerebella ...... 41
2.4 Constructs for transgenic mice ...... 42
2.4.1 L7AAUG add-back construct ...... 42
2.4.2 L7AAUG add-back-PKC construct...... 42
2.5 Preparation of mouse tail DNA ...... 43
2.6 Total RNA and mRNA isolation from mouse, rat or human tissue ...... 43
2.7 S’RACE of mouse and human L7 mRNA ...... 43
2.7.1 S' RACE of mouse L7 mRNA ...... 43
2.7.2 5'RACE of human L7 mRNA ...... 44
2.8 S' RACE of rat and human L7 mRNA ...... 45
2.8.1 3'RACE of rat L7 mRNA ...... 45
2.8.2 3'RACE of human L7 mRNA ...... 45
2.9 Making alpha-^P labeled DNA or RNA probes ...... 46
2.9.1 Making alpha-^P labeled DNA probe for EGFP
transgenic mice northern blot ...... 46
2.9.2 Making P-32 labeled DNA probe for human L7
northern blot...... 46
2.9.3 Making P-32 labeled RNA probes for EMSA and UV cross-linking..46
ix 2.9.3.1 Making full-length wt L7-3 UTR probe ...... 46
2.9.3.2 Making mutated L7-3 UTR probes ...... 47
2.9.3.3 Making RNA oligo probes ...... 48
2.10 Northern blot ...... 48
2.11 Electrophoretic mobility shift assay(EMSA) ...... 49
2.12 UV cross-linking of RNA-protein complex ...... 50
2.13 Making S-35 labeled probes for in situ hybridization ...... 50
2.13.1 Making L7 antisense probe ...... 50
2.13.2 Making SV40 3'UTR antisense probe ...... 51
2.13.3 Making GFP antisense probe ...... 51
2.13.4 Making human L7 antisense probe ...... 52
2.13.5 Making human CaBP antisense probe ...... 52
2.13.6 Making mouse exon 1A and IB specific antisense probes ...... 52
2.13.7 Making rat L7 antisense probe ...... '...... 53
2.13.8 Making rat CGI-145 antisense probe ...... 53
2.13.9 Quantitation of radioactivity of alpha-^S UTP labeled probe ...... 54
2.14 In situ hybridization ...... 54
2.15 Western blot ...... 57
2.16 Immunohistochemistry with fluorescent secondary antibodies ...... 58
2.17 Preparation of protein extract from wt or GFB4ransgenic animals ...... 59
X 2.18 Fluorescence measurement of GFP protein by fluorometer...... 59
2.19 Measuring the fluorescence of GFP protein using
fluorescence microscope ...... 59
2.20 Preparation of protein extract from mouse cerebellum ...... 60
2.21 Preparation of protein extract from rat cerebellum ...... 60
2.22 Purification of L7 3'UTR binding proteins from cerebellar protein
extract ...... 61
2.23 Concentrate protein samples by TCA precipitation ...... 64
2.24 SDS-PAGE of fractions and gel staining ...... 64
2.25 Sample preparation for in gel digestion and MALDI-MS ...... 64
3. Analysis of the 5’end of L7 mRNA in both mouse and human ...... 67
3.1 5’ Differential expression of two forms of L7 mRNA detected by
5’ RACE...... 67
3.2 L7 form A and form B mRNAs are detected both in cell body and dendrites
In younger animals ...... 69
3.3 Characterization of the rat L7 gene ...... 70
3.4 Cloning of the human L7 gene ...... 71
3.5 The human L7 mRNA is found in both the Purkinje cell body and dendrites
and the dendritic localization is developmentally regulated ...... 73
XI 3.6 Human L7 protein has two GoLoco domains as in rodent but has more
potential modification sites than in rodent ...... 74
3.7 Human L7 protein is specifically expressed in Purkinje cells ...... 75
4. I ransgenic approach for the study of L7 mRNA localization and
translation ...... 97
4.1 L7-3 UTR add-back construct ...... 97
4.2 Add-back-PKC construct...... 99
4.3 Characterization of GFP transgenic animals ...... 101
5. Biochemical analysis of the L7-3 UTR and purification of the binding proteins
CGI-145 and UNR ...... 123
5.1 The downstream half of the L7-3 UTR interacts with multiple cerebellar
cytoplasmic protein extracts ...... 123
5.2 Polyadenylation signal and its surrounding sequence are important for the
formation of RNA-protein complex ...... 125
5.3 Several proteins are involved in the RNA-protein complex ...... 127
5.4 Partial purification of protein components interacting with oligo3 ...... 128
5.5 Purification of the 50KD and 95KD L7-3’UTR-binding proteins from rat
cerebellar protein extract ...... 130
5.6 The 50KD protein is the rodent homologue of human CGI-145 homologue
and the 95KD protein is rat UNR ...... 133
xii 6 Discussion...... 194
6.1 The distribution of L7 transcripts in cerebellar Purkinje cells is
developmentally regulated ...... 195
6.2 Function of L7 protein ...... 197
6.3 Is the L7 3'UTR involved in the translocation of its message? ...... 200
6.4 The L7-3 UTR contains a cis-element that is conserved between
species ...... 203
6.5 L7-3’UTR-interacting proteins ...... 205
6.5.1 CGI-145 is ubiquitous and highly consenred ...... 205
6.5.2 UNR, an RNA binding protein, binds specifically to RNA with high
affinity ...... 206
6.5.3 UNR protein is involved in translation and mRNA decay, and is
essential for survival ...... 208
6.6 Conclusions...... 213
Reference List ...... 215
Mil LIST OF TABLES
Table
2.1 Table 2.1 human cerebellum tissues used In the experiments ...... 66
3.1 Amino acids composition in rodent and human L7 proteins ...... 96
XIV. LIST OF FIGURES
Figure
1.1 Schematic illustration of the structure of cerebellum ...... 38
1.2 An overview of trimeric G protein involved pathways ...... 39
1.3 Comparison of RGS, AGS and other GoLoco motif containing proteins 40
3.1 5'RACE of mouse L7 cDNA and L7 genomic sequence from
exon lA to exon 2 ...... 76
3.2 In situ hybridization of mouse L7 form A mRNA at different ages ...... 78
3.3 In situ hybridization of mouse L7 form B mRNA at different ages ...... 80
3.4 In situ hybridization of rat L7 mRNA and immunohistochemical staining
of rat L7 protein ...... 82
3.5 5'RACE of human L7 cDNA and human L7 genomic sequence
from exon 18 to exon 2...... 84
3.6 Comparison of human and rodent L7 gene structure ...... 86
3.7 In situ hybridization of mouse, rat and human L7 mRNA ...... 88
3.8 In situ hybridization of human L7 mRNA at different ages ...... 90
3.9 Alignment of human and rodent L7 proteins ...... 92
3.10 Detection of human L7 mRNA and protein by northern blot, western blot
and immunohistochemical staining respectively ...... 94
XV 4.1 Illustration of constructs for making transgenic mice ...... 105
4.2 Comparison of transgene mRNA localization in different
transgenic animals ...... 107
4.3 Illustration of GFP construct for generating transgenic mice ...... 109
4.4 GFP mRNA is specifically expressed in cerebellar Purkinje cells ...... 111
4.5 GFP protein is only expressed in cerebellum in tfie transgenic mice ...... 113
4.6 Protein extract made from GFP transgenic cerebellum showed
typical GFP protein emission property ...... 115
4.7 Quantitation of GFP protein in transgenic mice ...... 117
4.8 GFP aniami shows normal brain and cerebellum phenotype and GFP is
only expressed in cerebellar Purkinje cells under L7 promoter ...... 119
4.9 Purkinje cell marker proteins are normally expressed in GFP transgenic
mice and Purkinje cells showed normal phenotype ...... 121
5.1 Sequence of L7-3 UTR, RNA digos and mutated L7-3’UTR probes ...... 135
5.2 Oligo3 can compete equally well as full-length 3'UTR for the binding of
protein components ...... 137
5.3 APolyAm can form a similar RNA protein complex as wt 3'UTR does and it
can strongly compete with wt 3'UTR for the binding ...... 139
5.4 APolyAd forms a significantly reduced complex and could weakly compete
with wt 3'UTR for the binding...... 141
xvi 5.5 Polyadenylation signal surrounding sequence affects tfie binding activity.143
5.6 AE has no biniding ability and can hardly compete with wt 3'UTR 145
5.7 Polyadenylation signal AAUAAA sunrounging sequence is important for the
binding...... 147
5.8 Poly(A) and poly(U) strongly compete with wt 3'UTR for the binding 149
5.9 Wild type 3' UTR and oligo 3 strongly compete with the hot probe for the
binding of two protein bands on UV cross-linking gel ...... 151
5.10 Two protein bands were identified by UV cross-linking ...... 153
5.11 Assay of binding activities of rriouse OEAE fractions by UV cross-linking
and EMSA ...... 155
5.12 Protein profile of mouse OEAE fractions ...... 157
5.13 Both 90KD and 50KD proteins bind to heparin column and are
co-purified ...... 159
5.14 Protein profile of mouse heparin fractions ...... 161
5.15 EMSA of mouse HIC fractions ...... 163
5.16 Protein profile of phenylsepharose column fractions ...... 165
5.17 Binding activity assay of rat OEAE fractions by EMSA and UV
cross-linking...... 167
5.18 Protein profile of rat OEAE column fractions ...... 169
5.19 Binding activity assay of rat heparin fractions ...... 171
xvii 5.20 Protein profile of rat heparin fractions ...... 173
5.21 Activity assay of rat HIC fractions by EMSA and UV cross-linking ...... 175
5.22 Protein profile of rat HIC column fractions ...... 177
5.23 90KD protein was partially purified and excised for MALDI-MS ...... 179
5.24 Assay of binding activity of rat SP fractions ...... 181
5.25 Binding activity assay of rat mono Q column fractions ...... 183
5.26 Protein profile of rat mono Q Column fractions ...... 185
5.27 Propound search result of CGI-145 MALDLMS peptides ...... 187
5.28 Summary of CGI-145 MS/MS spectra ...... 188
5.29 In situ hybridization of CGI-145 in adult rat cerebellum ...... 189
5.30 Profound search of rat MALDI-MS peptides ...... 191
5.31 In situ hybridization of UNR in adult rat cerebellum ...... 192
XVIII ABREVIATIONS
3’ RACE - 3’ rapid amplification of cDNA ends 3' UTR - 3' untranslated region 5' RACE - 5' rapid amplification of cDNA ends 5’ UTR - 5’ untranslated region AGS - activator of G protein AN I - anisomycin AP-1 - activator protein 1 ATP - adenosine triphosphate Arc - activity-regulated cytoskeleton-associated protein BDNF - brain derived neurotrophic factor bp - base pair BSA - bovine serum albumin CaBP - calbindin CaMKIIa - Ca^"-calmodulin-dependent protein kinase lia subunit cDNA - complementary DNA cGMP - cyclic guanosine monophosphate CNS - central nen/ous system CPEB - cytoplasmic polyadenylation element binding protein CPSF - cleavage and polyadenylation specificity factor CRE - cAMP response element CSD - cold shock domain dCTP - deoxycytosine triphosphate DEP - Dishevelled/Egl-10/pleckstrin homology DEPC - diethyl pyrocaribonate DNA - deoxyribonucleic acid DTT - dithiothreitol EDTA - ethylenediaminetetraacetic acid EMSA - electrophoretic mobility shift assay FPLC - flow pressure liquid chromatography GAP - GTPase activating protein GDI - GDP dissociation inhibitor GDP - guanosine diphosphate GEF - guanine nucleotide exchange ktctor GFP - green fluorescent protein GoLoco - GOj/o-Loco GTP - guanosine triphosphate xix IRES - internal ribosomal entry site LTD - long term depression LTF - long term facilitation L IP - long term potentiation MALDI-MS - matrix assisted laser desorption ionization mass spectrometry MAP2 - microtubule-associated proteinZ MARTA - MAP2-RNA trans-acting proteins mCRD - major protein-coding region determinant NMDA - N-methyl-D-aspartate NP-40 - nonidet P-40 PABP - polyA binding protein PBS - phosphate buffered saline PCD - Purkinje cell degeneration PGP - Purkinje cell protein PGR - polymerase chain reaction PDZ - PSD95/Dlg/ZO-1 PH - pleckstrin-homology PINS - partner of inscuteable PMSF - phenylmethylsulphonyl fluoride PTB - phosphotyrosine binding PTB - polypyrimidine tract-binding protein RAR - retinoic acid receptor RARE - retinoic acid responsive element RGS - regulator of G protein signaling RNA - ribonucleic acid ROR - retinoic acid receptor-related orphan nuclear receptor RT-PGR - reverse transcriptase PCR SDS - sodium dodecylsulfate SDS-PAGE - sodium dodecylsulfate polyacrylamide gel electrophoresis SELEX - systematic evolution of ligands by exponential enrichment SSG - sodium chloride/sodium citrate SV40 - simian virus 40 TB-RBP - testis brain RNA-binding protein TBST - Tris-buffered saline Tween-20 TGA - trichloric acid TEA - Triethanolamine TPR - tetratrico peptide repeats UNR - upstream of N-ras UTP - uracil triphosphate ZBP1 - zipcode binding protein 1 XX Chapter 1
INTRODUCTION
Since our project is to study the cerebellar Purkinje cell specific gene L7, the introduction will start with a brief review of the structure of mouse cerebellum, followed by sections about the discovery, expression, regulation and functions of
L7 gene. Two topics related to the L7 functions will also be covered. One is the
RGS proteins and AGS proteins, the other one is the dendritic protein synthesis.
1.1 Cerebellum serves as an Ideal model for the study of the nervous system
During the past 100 years, many researchers have been attracted to the study of cerebellum because of its simple yet highly ordered structure (Altman and Bayer,
1997; Ghez, 1991; Voogd and Glickstein, 1998). In mammals, cerebellum is composed of three layers which contains only several types of neurons: Purkinje cells, granule cells, Golgi cells, stellate cells and basket cells (figure 1.1). The outermost layer is molecular layer which contains dendrites of Purkinje cells, axons of granule cells, stellate cells and basket cells. Beneath the molecular layer is a single layer of Purkinje cell bodies. Purkinje cells are the principal neurons of cerebellum. Their tree-like dendrites are aligned in the same plane
I and extend into the molecular layer toward the outer side of the cerebellar cortex.
Their axons, on the opposite side of the cell body, run into the white matter and
make contact with deep cerebellar nuclei, sending out the sole output information from cerebellum. The innermost layer is granule cell layer which contains a huge
population of small granule cells. The granule cell axons take an opposite
direction as the Purkinje cell axons, they pass the Purkinje cell layer and run into
molecular layer. In molecular layer, these axons normally bifurcate and run in
parallel with the surface of the lobules to form parallel fibers, parallel fibers then
form synapse with Purkinje cell dendrites. Dendrites from each Purkinje cell can
form synapses with many parallel fibers and receive input from them. Another
type of input to Purkinje cells comes from climbing fibers which originate from an
extracerebellar structure, inferior olive. Climbing fibers form synapses with
Purkinje cells both at their cell bodies and their dendrites. Apart from these two
types of direct input, Purkinje cells receive indirect input from mossy fibers which
originate from multiple sources outside cerebellum. Mossy fibers make synaptic
contacts with granule cell dendrites, and through granule cells, they send their
input to Purkinje cells. As shown in figure 1.1, these neurons and fibers form a
fabulous crystalline structure in the cerebellar cortex, and their lattice structure of
different layers and synaptic connections make cerebellum the most favored
anatomical model for the study of central nervous system (CNS). Another important aspect of cerebellum is the Information flow In this crystal-llke organization. Information coming from outside of the cerebellum either flows through climbing fibers to directly excite Purklnje cells or flows through mossy fibers by way of granule cells to excite Purklnje cells. The Information flowing In the latter route also excites stellate cells, basket cells and Golgl cells, but these cells will then inhibit Purklnje cells. Information leaving cerebellum can only convey through axons of Purklnje cells which will pass on an Inhibitory Input to their target. On one hand, when an excitatory Input Is received by Purklnje cells from climbing fibers or from parallel fibers, an Inhibitory Information will then be sent out of the cerebellum. On the other hand, when Inhibitory Information reaches Purklnje cells from basket cells, stellate cells and Golgl cells, an information to release inhibition will be conducted to the target of Purklnje cells.
This delicate information circuitry makes cerebellum an Instructive model for understanding the network of Input and output In CNS.
Apart from these structural properties, the expanding new hypotheses of cerebellar functions have also shown the Importance of the study of cerebellum.
The growing number of data have suggested that cerebellum Is not only part of the essential motor control system (Ghez, 1991), but also Is Important for learning (Thompson et al., 1997), timing (Ivry and Keele, 1989; Keele and Ivry,
1990), sensory acquisition (Bower, 1997; Gao et al., 1996), attention (Allen etal.,
1997), problem solving, error detection, language (Kawato and Gomi, 1992; Leiner et al., 1991; Leiner et al., 1993), emotions (Schmahmann and Sherman,
1998), etc. So, from any aspect stated above, the fascinating well organized anatomical structure, the fine Information pathways or the diverse functions, cerebellum has presented Itself as an Ideal model for the study of CNS.
1.2 L7/pcp-2 is specifically expressed in cerebellar Purklnje cells
1.2.1 Discovery of L7 gene
In an effort to discover cell-type specific gene expresslolns which are Important In regulating the development of cerebellum, one Purklnje cell specific gene was
Identified by two labs. By differential hybridization using wild type and
Lurchei{Lc) cDNA probes, Oberdick et al. was able to Identify a Purklnje cell specific clone L7 (Oberdick et al., 1988). Similarly, by subtractive hybridization between wild type and Purklnje cell degeneration (pcd) mice, Nordqulst et al obtained a PCD-5 clone whose product Is the same as L7 gene product, and
PCD-5 was mapped to chromosome 8 In mouse (Nordqulst et al., 1988). This gene was later named L7/pcp-2 (pcp-2 stands for Purklnje cell protein
2(Vandaele et al., 1991). L7/pcp-2 gene (henceforth called L7) has been shown to be expressed exclusively In cerebellum and In eyes by northern blot. In cerebellum, Purklnje cell Is the only cell type that expresses L7. L7 mRNA was shown to have a size of -510 to -600nts Including polyA tall by northern blot and
by primer extension. Within this sequence, a predicted ORF encodes a 99-amlno
acid protein with a molecular weight of 11KD. The presence of L7 protein In cerebellar Purklnje cells was confirmed both by western blot and by immunohlstochemistry using L7 antisemm. The expression level of L7 protein is close to that of actin and this makes it a major component in Purkinje cells. In eyes, L7 protein was found to be localized in retinal bipolar neurons by immunohistochemistry (Berrebi et al., 1991). Although cerebellar Purkinje cells and retinal bipolar neurons do not have any immediate lineage relationships, they showed several common properties. The exclusive expression of L7 protein in both cell types is one of the properties they share.
1.2.2 L7 expression in cerebellum Is regulated spatially and temporally, aiKi
L7 promoter is a valuable cell type specific expression tool
To further investigate the cell-type specific expression of L7 and to provide a powerful marker for Purkinje cells and for retinal bipolar neurons, different versions of transgenic mice bearing 0-galactosidase transgene driven by L7
promoter were generated. L7 gene was shown to have four exons contained in a
2kb genomic sequence. Oberdick et al. inserted a 3kb LacZ gene in frame into the fourth exon of L7, this construct was expected to make a fusion protein of L7
and p-galactosidase. This constoict also contains 4kb fragment upstream of
transcription start site and 2kb fragment downstream of the polyadenylation
signal. Among all the transgenic animals they obtained, p-galactosidase activity
was detected in both cerebellar Purkinje cells and in retinal bipolar neurons. This
expression pattern of p-galactosidase faithfully recapitulated the the expression of wild type L7, indicating sequence elements needed for cell-specific expression are present in the construct (Oberdick et al., 1990). Using the same transgenic animal, Smeyne et ai. showed that the expression of p-galactosidase is in parasagittal bands of Purkinje cells in younger animals. After P2, Purkinje cells between these bands start to express g-galactosidase and by P9 no banding pattern can be seen because all Purkinje cells express the p-galactosidase
(Smeyne etal., 1991).
In another work, Vandaele et al. generated two constructs by replacing L7 coding region with the LacZ gene. The difference of the two constructs lies in the 5’ end sequence. The shorter construct has only 0.4kb of 5’ end upstream DNA while the longer construct contains 3.5kb upstream sequence. For both constructs, sequence from the start codon in exon 2 to the stop codon in exon 4 was substituted by LacZ and their 3' ends have only 0.4kb flanking sequence. None of the transgenic animals bearing either construct expresses p-galactosidase in retinal bipolar neurons suggesting that the sequence responsible for this expression is missing in their constructs. This missing sequence could be in the
5’ or 3’ end flanking sequence or could be in the replaced sequence in the middle of the gene. The expression pattern of the LacZ varied from line to line for these two constructs. But one obvious result is that the longer construct gave more restricted LacZ expression pattern and p-galactosidase activity was always detected in Purkinje cells. This suggests that the extra 3.1 kb 5' flanking sequence in the longer construct contains regulatory element that shapes the expression pattern. Even in the longer construct bearing animals, ectopic expression was detected In several sites. This suggests that the 5’ upstream sequence by itself is not sufficient for the specific expression of transgene in
Purkinje cells, and sequence either in the replaced region or downstream of the gene is needed. In the longer construct, they also noticed that the LacZ expression in Purkinje cells showed parasagittal compartments. Transgenic animals bearing short constmct displayed a wide variety of expression pattern of
(3-galactosidase. This clearly showed that removing of the DNA from the flanking region or internal region severely affected the expression specificity of the L7 promoter (Vandaele et al., 1991).
In another elaborate work, Oberdick et al. used the same L7-LacZ fusion construct and made deletions of the 5’ end upstream region to examine the expression pattern and the regulatory elements controlling the expression
(Oberdick et al., 1993). What they showed is that as little as 0.25kb of S' flanking
sequence is sufficient to keep the Purkinje cell specific expression, but the
truncated promoter caused a decreased expression of p-galactosidase in adult
animal. They found that an inhibitory element is present between -0.5 and -I.Okb which represses the |3-galactosidase expression during embryonic and perinatal
stage. They also showed that p-galactosidase expression in lateral parasagittal
bands was repressed in 4kb or Ikb promoter constructs but elevated in truncated promoter constructs(0.5kb and 0.35kb). Purkin^ cells between the bands quickly express p-galactosidase in 4kb and Ikb promoter constructs after P2, and no banding pattern can be seen after P9. But with truncated promoter, especially the
0.35kb promoter, the expression oTp-galactosidase in the Purkinje cells between the stripes is delayed after P2, and the banding pattern can still be seen at P30.
This suggests that an enhancer which promotes the expression of g - gaiactosidase in all Purkinje cells after P2 is present in the L7 5’ upstream DNA.
Their results indicate that the expression of L7 in cerebellar Purkinje cells is controlled by multiple elements in the 5' upstream sequence, these elements could be positive or negative, and could regulate the expression spatially or temporally. When this result is compared to Vandaele’s, elements responsible for the cell-type specific expression in cerebellum should be present in the 2kb fragment replaced by LacZ in Vandaele’s construct. Taken together, as a major protein in Purkinje cells, the expression of L7 is regulated by elements
responsible for cell-type specificity, by elements responsible for banding pattern
and by elements responsible for time course. Based on this knowledge, L7
promoter has been successfully used for driving different transgenes to
specifically express in cerebellar Purkinje cells (De Zeeuw et al., 1998). 1.3 The expression of L7 is regulated by thyroid hormone and other fectors
1.3.1 RORa regulates L7 expression
One protein factor found to affect the expression of L7 gene is RORa which belongs to the nuclear hormone -receptor superfamily. It has been shown that in staggerer{sg) mice which carry a deletion within the RORa gene, the expression of L7 is not detectable (Hamilton et al., 1996). RORa is a retinoic acid receptor-
related orphan nuclear receptor. In mouse L7 gene, an ROR response element was found in the proximal promoter region, it has a well conserved sequence of
GTMCIGGGTCA. The bold type letters are the core sequence, the underlined
I at -1 position and A at -4 position are also identical to the consensus sequence
required for high affinity binding. L7 gene ROR response element is not part of
any known dimeric response element, but it is also recognized by COUP-TF at
high affinity (Anderson et al., 1998). Like L7 protein, members of ROR family are
expressed both in cerebellar Purkinje cells and in retinal bipolar neurons(Becker-
Andre et al., 1994; Nakagawa et al., 1997). In vitro experiment showed that
expression of L7 gene is activated by RORa, which binds as a monomer to the
ROR responsive element in the promoter. L7 expression has also been shown to
be activated by retinoic add receptor (RAR), which binds to a retinoic add
responsive element (RARE) in the promoter region. It has been demonstrated
that RORa-mediated induction of L7 expression is further activated
synergistically by RAR. So both RORa and RAR were suggested to regulate the
expression of L7 gene (Matsui, 1997). 1.3.2 Evidence of other L7 gene regulators
Other potential cis-elements identified by sequence analysis in L7 gene include cAMP response element (CRE), AP-1 binding sites and Oct protein binding
(Vandaele et al., 1991). Within the proximal promoter region, an AT-rich fragment and a consensus E-box were identified by DNA footprinting (Oberdick et al.,
1993). The AT-rich fragment has three TAAT repeats wherein provides a conserved binding site for homeobox protein and POU domain transcription factors. By mutating this AT-rich fragment, it was shown in transgenic animal that the expression pattern of L7 protein in cerebellar Purkinje cells was dramatically changed. And this indicated that the binding of potential homeodomain containing transcription factor to this AT-rich region is important for the normal expression of L7 gene in Purkinje cells. There is also evidence to show that L7 gene contains thyroid hormone response elements and is regulated by thyroid hormone (Zou et al., 1994) and COUP-TF (Anderson et al., 1998). In brief, the expression of L7 in cerebellar Purkinje cells is a complex process. It is regulated by multiple factors, including nuclear receptor factors, especially RORa, homeodomain transcription factors, POU domain transcription factors and others.
The regulation by these factors would affect the expression level, expression pattern and expression time. It’s a well organized, fine tuned process.
10 1.4 L7 protein Interacts with Go, and Goo modulating GDP release from Go
1.4.1 L7 knockout animals showed no significant phenotype
Since the cloning of the L7 gene, Investigators have been trying to determine the
function of its product. The sequence of L7 protein was analyzed for any
homology to other known function proteins (Oberdick et al., 1988), but no
obvious clue was obtained at that time. Because L7 gene is expressed at a high
level during an important developmental period of the cerebellum, and also
because its expression is highly regulated demonstrated by different factors, one
would hypothesize that its function may be critical for the development of
cerebellum. This hypothesis led to the generation of L7 null mice (Mohn et al.,
1997; Vassileva et al., 1997). To our surprise, L7 null mutant animals show no
significant difference when compared to their wild type littermates. Histological
experiments showed that L7 null mutant mice had similar body weight, brain
weight and cerebellum weight to that of wild type animals. Mutant animals had a
similar number of Purkinje cells in the cerebellum, similar depth of molecular
layer and similar cellularity of the molecular layer. Purkinje cells showed no
aberrant morphology of their cell body, axons and dendrites in the mutant
animals. And also no subcellular structure changes were noticed in the mutant
animals. Behavioral studies were not able to detect any defldts In L7 knock-out
mice either. The explanations for the phenotype of L7 knock-out animal are; 1)
L7 protein plays little role in the Purklnje cells. However, as we are going to show
in chapter 3, L7 protein Is highly conserved between rodent and human, and this
II suggests a conserved function in these species, 2) L7 protein plays a role that is not easily detected by the methods used in the mentioned literature, 3) or L7 function in L7 null mice was salvaged by other molecules which have similar functions. The choice of these possibilities has recently been clarified by analysis of L7 knockout mice using an accelerating rotarod (Gebhardt and Oberdick, unpublished data). In contrast to the expected result, L7 knockout animals were found to have an improved perfonnance by this assay, suggesting a role for L7 in signal dampening or homeostasis.
1.4.2 L7 protein interacts with Go subunit
Heterotrimeric G proteins are part of a complex membrane signaling system used for transmitting extracellular signals into the cell. They are coupled to seven-transmembrane receptors. These G proteins are consisted of a, p, and y subunits. Go subunit contains the binding site of GTP and GDP. In the resting state, GDP-bound Go and Py subunits form a trimeric protein. Upon the stimulation of the coupled receptor, Go exchanges GDP for GTP and Py subunits are released from the a subunit. In this state, Go subunit can regulate adenylyl cyclase, phospholipase Cp, 1C and Ca^* channels and cGMP phosphodiesterase. Py subunits form a dimmer and are anchored to the membrane. Py dimmer also plays important roles in signal transduction, it can regulate 1C channel, phospholipase Cp and some adenylyl cyclase and so on.
The transition between the GDP-bound trimeric inactive state and GTP bound
12 active state is regulated by different factors. Binding of GAP(GTPase-activating protein) to Go subunit accerates the hydrolysis of GTP, and binding of GDI(GDP dissociation inhibitor) to the Ga inhibits GOP release(Marinissen and Gutkind,
2001)(figure 1.2). L7 protein has shown to function as such a protein to regulate the release of GDP from Go.
The first hint for the in vivo function of L7 protein came from a yeast two hybrid experiment in which Goo was used as bait (Luo and Denker, 1999). In this study
L7 was found to be a binding partner of Goo. It was shown that GOo and another pertussis toxin-sensitive family member, Goe, could interact with GST-L7 fusion protein in a pull down experiment. But GOs, a member of the cholera toxin- sensitive family, could not interact with GST-L7 fusion protein. In an in vitro cotransfection assay, L7 was coimmunopredpitated with Goo. Kinetic analysis showed that L7 protein could activate GDP release from the G protein and functioned as guanine nucleotide exchange factor (GEF). These findings suggested that L7 protein could senre as a G protein modulator by activating
GDP release. In another study (Natochin et al., 2001), Natochin et al. showed that L7 protein had a notably higher affinity for Goi than Goo, but no detectable
affinity to Gog. This is consistent with the previous report. They also showed that
L7 protein preferably bound to GDP-bound form of Go, and GOo and it inhibited
the spontaneous binding of GTPyS to Go, and Goo. In sharp contrast to the other
study, L7 protein displayed inhibition of GDP release from Goi and GOo in this
13 study. These two studies share a common conclusion that the L7 protein Is involved in the regulation of Goi and Goto, but not Got,. They have one major difference in whether L7 protein Inhibits or activates the dissociation of GDP from the Go subunits. One known fact that may contribute to this difference Is that the
L7 constructs used for In vitro translation In their studies are different. The L7 construct used by Luo et al has extra amino acids at Its N terminal compared to the one used by Natochin et al. Also as mentioned by Natochin et al, the GST-L7 fusion protein used by Luo et al has a mutation which could possibly abolish the function of L7 protein. As we will discuss later some other G protein modulators which are related to L7 exhibit Inhibitory effects on GDP release from Go subunits.
1.5 L7 protein contains GoLoco motif
Recently, a new protein motif, GoLoco (or GOi/o-Loco) motif, has been Identified
by comparing mammalian RGS proteins to Drosophila Loco protein (Granderath
et al., 1999). The GoLoco motif contains 19 amino acids which has a consensus
sequence of EELLEMLxxxQSxRMEEQR. The Q residue at position 11 and QR
residues at positions 18 to 19 are Invariant residues. So far, several proteins
have been shown to contain GoLoco motlf(s). These proteins Include three RGS
proteins and several other G protein modulators. GoLoco motlf-contalning
proteins have been Identified In C .elegans, Drosophila, mouse, rat and human.
In the 99-amlno-acld L7 protein, one GoLoco motif has been found that It has a
14 sequence of DNLMDMLVNTQGRRMDOQR with an identity of 52% and an similarity of 73% to the consensus sequence. Since L7 protein and several other
GoLoco motif-containing proteins displayed similar interactions with Go, and Goo subunits (Natochin et al., 2001), and since some G protein modulators, especially some RGS proteins and AGS proteins, also contain GoLoco motif (Granderath et a!., 1999; Mochizuki et al., 1996; Snow et al., 1997), it is very likely that the L7 protein has a similar function as some of the RGS and AGS proteins.
1.6 RGS proteins belong to a protein family and undertake multiple functions including GAP and GDI
With the study of G protein modulators, a family of GTPase-activating proteins
(GAPs) called regulator of G-protein signaling (RGS) proteins were defined based on sequence homologies (De Vries et al., 1995; Druey et al., 1996; Koelle and Horvitz, 1996). The most important feature of RGS proteins is that they share a homologous 120-amino-acid RGS domain which is responsible for the binding of RGS proteins to Ga subunits (De Vries et al., 1995). Some RGS proteins also contain additional domains to provide other functions or crosstalks to other players in the signal network (De Vries and Gist, 1999).
15 1.6.1 RGS proteins are stemmed from yeast Sst2 protein and are present In other lower eukaryotic organisms
RGS proteins have been Identified in many organisms, ranging from S. cerevisiae to human (De Vries et al., 2000b). These proteins have evolved from the S. cerevisiae Sst2 protein which was shown to interact with the pheromone receptor-linked Go subunit, Gpai (Dohlman et al., 1995; Dohlman et al., 1996).
Sst2 protein attenuates G protein signaling by accelerating GTP hydrolysis and hence desensitizes the response to a-factor pheromone (Chan and Otte, 1982b;
Chan and Otte, 1982a). In Aspergillus nkiulans, an RGS protein RbA has been shown to be necessary for mycelial proliferation and asexual sporulation (Yu et al., 1996). It displayed an important function in balancing growth and sporulation by regulating FadA, which is the a subunit of G protein. Genetic analysis showed that FIbA exhibited negative regulation of FadA, very likely through activating the
GTPase activity of FadA, a similar way used by Sst2 in yeast.
Egl-10, a C. elegans protein, is among the first RGS proteins identified (Koelle and Horvitz, 1996). It inhibits the egg-laying and locomotor behavior by inhibiting the signaling of GOA-1, a homologue of the G proteins Go (Dong et al., 2000).
An homologue of Egl-10, dRGS7, was identified in Dmsophila (Elmore et al.,
1998). Apart from the RGS domain, dRGS7 protein also has a domain which is homologous to dishevelled and pleckstrin, implying that it can also interact with other signaling components. Another gene identified in Drosophila that encodes
16 an RGS protein is loco (locomotion defects) (Granderath et al., 1999). loco gene can give rise to two different transcripts which differ in their 5' ends. These two transcripts in turn will be translated into Loco-cl and Loco-c2. They share the same C-terminal sequence, but Loco-c2 has extra 346 amino acids at its N- terminal. Loco-cl mRNA was found in the glial cells in the CNS, but Loco-c2 mRNA could not be detected in CNS. Besides the RGS domain. Loco protein also has one Goloco motif at its C-terminal. It has been shown that Loco protein interacts with Go, and is required for the glial differentiation. Experimental evidence indicated that the interaction between Loco protein and Goi is not only mediated by RGS domain but also by Goloco motif. And this finding supports the idea that the GoLoco motif is important for the interaction or regulation of G protein by GoLoco motif containing proteins.
1.6.2 Mammalian RGS proteins are grouped into subfemilies
In mammalian systems, a series of RGS proteins have been identified in mouse, rat and in human. Different RGS proteins have been grouped into different subfamilies mainly based on their structural organizations. So far, about 6 subfamilies have been identified. Only members from one subfamily contain
GoLoco motif. But it is instructive and helpful to have an overview of all the subfamilies, so that one can easily compare the structure and function of these
RGS proteins, especially to compare the GoLoco motif-containing RGS proteins to those without it.
17 The First subfamily contains GAIP, RGSZ1, RET-RGS1 and RGS17. By yeast two hybrid screening, De Vries et al. Identified human GAIP (G Alpha Interacting
Protein) and showed that it specifically interacts with Goia mediated by its RGS domain (De Vries et al., 1995). A unique cysteine string was found upstream of the RGS domain in GAIP indicating that it is substrate of palmitoylation
(Gundersen et al., 1994). RGSZ1 is a major GAP specific for GCz in brain (Wang et al., 1997). It has an unusual selectivity for Goz among RGS proteins and it is tightly associated to the plasma membrane (Wang et al., 1998). It also contains the cysteine string. RET-RGS1 is an RGS protein that is specifically expressed in
retina. Apart from the GRS domain at its C-terminus, it has a transmembrane
region and a stretch of 9 cysteines. RET-RGSI is an integrated membrane
protein. The in vitro expressed RGS domain of RET-RGSI can activate the
hydrolysis of GTP by transducin (Faurobert and Hurley, 1997). Further analysis
on the RET-RGS showed that it participated in retina specific synaptic
transductions (Faurobert et al., 1999). RGS17 was isolated during a yeast two
hybrid screening, it has one RGS domain and also an upstream cysteine stretch
(Jordan et al., 1999). The characteristic cysteine string apart from the RGS
domain in these RGS proteins is a predicted palmitoylation site. Some of the
GAIP has been shown to be membrane associated (Fischer et al., 2000), while
RGSZ1 and RET-RGSI are membrane proteins. Functionally, GAIP, RGSZ1,
and RET-RGSI negatively regulate the G protein coupled pathway by a similar
18 mechanism that is to accelerate the hydrolysis of GTP (Wang et al., 1998), and
RGS17 is expected to have a similar function as other members.
The second subfamily contains most of the members, RGS1,2,3 4,5,8,13 and 16.
RGS1 was isolated by a subtractive cloning from phorbol ester-stimulated human
B lymphocytes (Hong et al., 1993). it is a Go, and Goq GTPase activating protein and it has been shown to Impair the Goi mediated response of B lymphocytes
(Moratz et al., 2000). RGS 2 was Isolated from blood mononuclear cells treated with cycloheximide (SiderovskI et al., 1994). RGS3 was originally Identified by screening a B cell cDNA library using a probe homologous to the sequence consen/ed in RGS1 and RGS2 (Druey et al., 1996: Gold et al., 1997). It has two isoforms (Mittmann et al., 2001) and has been shown to be a GTPase activating protein for Goi and Gcq (Scheschonka et al., 2000). RGS4 is exclusively expressed in brain and its crystal structure has been solved (Berman et al., 1996;
Druey et al., 1996; Gold et al., 1997; Tesmer et al., 1997). RGS5 has been found
to bind to Gdi.GOo and GOq. but not Go, (Zhou et al., 2001). RGS8 Is a neural-
tissue-specific protein. It preferentially interacts with GOo and Goia and functions
as a GAP (Saitoh et al., 1997). RGS13 has been found In most part of the brain
(Grafstein-Dunn et al., 2001). RGS16 was Identified to be expressed In liver and
pituitary, and it binds to Goi and GOo (Chen et al., 1997). These RGS proteins
only have one RGS domain, a short N terminal and C terminal flanking sequence
with the exception of RGS3, which has a long N terminal sequence. Another
19 common feature they share is the subfamily specific conserved residue. For this subfamily, a conserved Ser residue can be found at the same position in all family members (Zheng et al., 1999).
RGS6,7,9 and 11 form another subfamily. In addition to the C terminal RGS
domain, these RGS proteins all have the same two additional domains. DEP
domain, which has a Dishevelled/Egl-10/pleckstrin homology, is situated at the N
termini of the proteins. GGL domain, which has homology to Gy, is located in the
middle of the peptide and is very close to RGS domain. Each member in this
subfamily has multiple isoforms of mRNA due to altemative splicing, and some of
these isoforms will give rise to different protein products (Snow et al., 1999;
Zhang et al., 1999b). All of these four RGS proteins in this subfamily have been
shown to interact with Gp5 subunit to form an RGS/GPS complex and in turn act
as GAP of Go. The interaction between RGS and Gp5 is mediated by the GGL
domain (Cabrera et al., 1998; Cowan et al., 2000; He et al., 1998; Posner et al.,
1999; Snow et al., 1998b). The DEP domain is likely responsible for the
localization of these RGS proteins to the membrane, a function of Dishevelled in
Drosophila (Axelrod et al., 1998). Egl-10 of C. elegans also belongs to this
subfamily.
The three subfamilies discussed above cover most of the mammalian RGS
proteins identified so far. Other subfamilies contain only two to three identified
2 0 RGS proteins for each. The subfamily whose members contain GoLoco motif is the RGS12/RGS14 subfamily. RGS12 and RGS14 were isolated together and showed more homologies to each other than to any other RGS members (Snow et al., 1997). RGS 12 itself has many different isoforms due to the altemative splicing (Chatterjee and Fisher, 2000; Snow et al., 1998a). In the longest version of RGS 12, in addition to the RGS domain, it has several other domains and motifs. The RGS domain is in the middle of the RGS12 protein. Upstream of the
RGS domain, it has one PDZ(PSD95/Dlg/ZO-1) domain and one
PTB(phosphotyrosine binding) domain. Downstream of the RGS domain, it has one Raf-like region, one GoLoco motif and one coiled-coil domain. RGS14 is much smaller than RGS12. It has an RGS domain, a Raf-like region and a
GoLoco motif organized in a similar manner as in RGS12. It has been indicated that RGS 12 is a GTPase-activating protein for Go;. The PTB domain in RGS12 has been shown to mediate a direct interaction between RGS12 and a tyrosine- phosphorylated calcium channel (Schiff et ai., 2000). The presence of a PDZ domain in RGS12 would mediate protein-protein interactions and localize enzymatic activities or other domains to the submembrane region (Ponting et al.,
1997). RGS 14 is a GAP for Goi and it inhibits interleukin-8 receptor-mediated
MAP kinase activation (Cho et ai., 2000). It also interacts with Goo. but not GOs and GOq. RGS 14 also displayed an ability to bind to Rap proteins through its Raf- like region (Traver et al., 2000). One unique property of these two proteins is that they both contain a GoLoco motif. More evidence about the interaction between
21 the Ga subunit and the GoLoco motifs In RGS12 and RGS14 will be discussed later. There are another two subfamilies. One of them Includes Axin (Ikeda et al.,
1998; KIshida et al., 1998; Zeng et al., 1997) and conductin (Behrens et al.,
1998) and the other one Includes pi 15-RhoGEF (Hart et al., 1998; Kozasa et al.,
1998), Lsc (Whitehead et al., 1996) and PDZ-RhoGEF (Fukuhara et al., 1999).
Both Axin and Conductin have glycogen-synthase-klnase-30-blndlng domain, 0- catenin binding domain and DIX domain (Dishevelled and axin homology domain) in addition to the RGS domain, pi 15-RhoGEF, Lsc and PDZ-RhoGEF have both a Dbl-homology domain and a pleckstrln-homology domain In addtltion to an RGS domain. PDZ-RhoGEF also has a PDZ domain at Its N terminus. The
RGS domain acts as a GAP while the DM domain Is a GDP-GTP exchange factor for the small GTPase Rho. Some G protein receptor kinases also contain RGS domain and form another subfamily. Several RGS proteins have not been classified into any of these subfamilies, like D-AKAP2 (Huang et al., 1997), a
PKA anchoring protein.
At this time, the knowledge about the function of RGS proteins has expanded from merely a GAP protein. They could be a direct antagonists of Go effectors
(Hepler et al., 1997; Tesmer et al., 1997) or they could act as scaffold proteins
(Ikeda et al., 1998) . The RGS domaln-flanking domains or motife allow RGS proteins to be associated with membranes (like RET-RGSI), or to mediate protein-protein interactions (like PDZ domain), or to anchor protein kinase to Its
2 2 target and so on. The study of RGS protein has also started to unveil the mechanism behind some diseases (Behrens et al., 1998; Buckbinder et al., 1997;
Ingi et al., 1998; Kim et al., 1999; Kishida et al., 1998; Roush, 1996).
1.7 AGS proteins
AGS(Activator of G protein Signaling) proteins were isolated using a S. cerevisiae expression cloning system designed to detect mammalian activators of the pheromone response signaling in the absence of GPCR(G protein coupled receptor). Three AGS proteins have been identified. AGS1 is a Ras-related G protein which facilitates GTP exchange on Go (Cismowski et al., 1999). AGS2 is a homologue of mouse Tctexi, which is light chain component of dynein
(Takesono et al., 1999). AGS3 was isolated together with AGS2, and later it was found that AGS3 has two different forms (Pizzinat et al., 2001). The longer form has two functional domains. At its N terminus, it has seven TPR (tetratrico peptide repeats) sequences, while at its C terminus, it has four GoLoco motifs.
This form of AGS3 is highly enriched in the brain and has been shown to specifically interact with Gau. 3, but not GOs and Goq, especially with GDP-bound conformation (Bernard et al., 2001; De Vries et al., 2000a). Another shorter form
of AGS3 has been found enriched in the heart. This shorter form AGS3 does not
have the N-temninal TPRs, it has three complete GoLoco m otik and a portion of
the fourth GoLoco motif found in the longer form. The Short form of AGS3 also
specifically interacts with GDP-bound Gog. GST-AGS3-short has been shown to
23 bind to GoiiÆ, Goja, but not Ga® and Goq. This finding Is consistent with the evidence that the TPR domains In AGS3 do not Interact with Gon/z (Bernard et al., 2001). In some other studies, the TPR domain has been Indicated to be
involved in protein-protein Interactions (Blatch and Lassie, 1999).
1.8 Other GoLoco motif-containing proteins
Several other proteins outside the RGS and AGS protein families also contain
GoLoco motifs. One of them is the human mosaic protein LGN. It got Its name
LGN because It has 10 LGN repeats at Its N terminal half (Mochizuki et al.,
1996). LGN has four GoLoco motifs at its C terminal and It has been shown to
interact with Goiz. RaplGAP is a GTPase activator of the monomeric G protein
Rapi (Rubinfeld et al., 1991). It has one GoLoco motif at Its N terminus and a
Rap_GAP domain. RaplGAP has been shown to Interact with Goi (Mochizuki et
a!., 1999) and Goz (Meng et al., 1999). PINS(partner of Inscuteable) was
isolated from Drosophila (Yu et al., 2000). It Is expressed both In epithelial cells
and in neuroblasts. It has a similar structure as AGS, seven TPR motifs at the N
terminal and three GoLoco motif at the 0 terminal. Evidence support that PINS Is
involved in the process of cell polarity. The Interaction between PINS and Go has
also be observed (Schaefer et al., 2000). Some other proteins have also been
suggested to contain GoLoco motlfe (SiderovskI et al., 1999). A comparison of
RGS, AGS and other GoLoco motif containing proteins has been illustrated in
figure 1.3.
24 1.9 GoLoco motif Is the interaction site for Go subunit and it acts as GDI
As stated above, GoLoco motif-containing proteins have been identified In different protein families, which may have different functions and different mechanisms behind the function, but they all share common property of interaction with the Ga subunit of the trimeric G protein. More and more evidence clearly shows that the GoLoco motif Is responsible for the binding of these proteins to the Go subunit For example, by splitting the AGS3 protein Into the N terminal TPR half and the C terminal GoLoco half, it was shown that only the C terminal GoLoco half had the ability to Interact with Gai. The N terminal GoLoco half of AGS3 also showed the ability to bind multiple Ga subunits (Bernard et al.,
2001). Natochin and colleagues used GoLoco motlf-contalning peptides from
AGS3 to show that the GoLoco motif can Inhibit the binding of GTPyS to GoU and also can reduce the rate of GDP release from Go subunit This suggests that
GoLoco motif Is a GDP dissociation Inhibitor (GDI). They further confirmed this finding by showing that GoLoco motif-containing AGS peptides could effectively suppress the hydrolysis rate of the GTP by Goi, because this Is an Indication of the GTP-GDP exchange rate (Natochin et at, 2000). Also, the peptide which contained all four GoLoco motifs was more effective as a GDI than the one with only the last two GoLoco motik. In an Independent study. De Vries and colleagues confirmed this finding and suggested that the last GoLoco motif In
AGS3 is a major contributor for the binding of C3o subunit (De Vries et at, 2000a).
In another study, Peterson et al. analyzed the GoLoco motif primary structure
25 with helical wheel and Chou-Fasman methods to show that this motif is capable of existing as an amphipathic helix. In this helix, F4, A8, Q11, MIS and R19 residues are located at the side of the helix and form a hydrophobic sector. They proposed that this hydrophobic sector is important for the binding of Go subunit
(Peterson et al., 2000). In one case, when the R19 was replaced by F, AGS3 lost the binding activity, indicating the importance of this sector (Takesono et al.,
1999). When short peptides were synthesized according to the sequence of four
Go Loco motifs in AGS3 and used to compete with AGS3 for the binding to the
Gqi subunit, these peptides completely blocked the binding of AGS3 to Goi and inhibited the binding of GTPyS to the Goi. On the other hand, the R19F mutant peptide couldn’t compete with the binding and couldn’t inhibit the binding of
GTPyS to the AGS3. Wild type GoLoco peptides also exhibited the ability to stabilize the GDP-bound conformation by reducing GDP dissociation rate, and the R19F mutant peptide lost this ability. All these findings support the idea that the GoLoco motif is a GDI.
In an recent study, Kimple and colleagues showed that GoLoco motifs in RGS12 and RGS14 specifically bound to Goiw in the GDP-bound conformation in a yeast two hybrid system. When an SPR real time binding assay was used to determine the interaction between the Ga subunit and full length RGS or GoLoco
motif peptide, GoLoco motif displayed an ability to bind to the GOP-t)ound Goi whereas RGS box showed an ability to bind to both GDP-bound Goi and Goo. A
26 fluorescence-based real time GTPyS binding assay showed that GoLoco motifs
In both RGS12 and RGS14 are GDI, and these motifs stabilize the GDP-bound
GOi and keep it in an inactive state. This finding is consistent with the one found
In AGS3 (Kimple et al., 2001).
Natochin et al. also showed that the GoLoco motifs in LGN and
RaplGAPdlsplayed an ability to interact with Goi. And these motifs inhibited the
GTPyS binding to Goi. By monitoring the rate of GDP release, they showed that
GoLoco motifs in these two proteins served as GDI.
So far, all of the GoLoco motifs examined showed an ability to interact with GDP-
bound GOi, whereas Luo et al also showed that the GoLoco motif in L7 protein
could Interact with Goo (Luo and Denker, 1999). The most important discovery
about the GoLoco motif is the finding that GoLoco motif act as GDP dissociation
Inhibitor, although Luo et al argued that GoLoco motif in L7 activates GDP
release. In RGS12 and RGS14, RGS box functions as GTPase activating protein
to accelerate the hydrolysis of the GTP associated with Goi and Goo, while at its
C terminal, GoLoco motif inhibits the release of GDP from Go,. Between these
two functional domains is the Raf-like region. This suggests that a crosstalk
between the trimeric G protein pathway and monomeric G protein pathway could
be Involved In the RGS12 and RGS14 mediated signaling. Furthermore, in
RGS12, the N terminal PDZ domain and PTB domain can also crosstalk with the
27 player in other pathway. In the AGS3 proteins, the N terminal TPR domain may act as a scaffold and bring other proteins together, while the C terminal GoLoco motifs interact with the trimeric G protein pathway. One could expect that crosstalk between trimeric G protein and other pathways would happen when
TPR binds other proteins. When we compare the data from RGS12, RGS14 and
AG S3 to L7, Natochin et a! showed a similar result that the GoLoco motif in L7 protein acts as GDI (Natochin et al.. 2001). In contrast, Luo et al. showed an opposite conclusion (Luo and Danker, 1999). In Natochin’s paper, they found that the L7 construct used by Luo et al. had a mutation in the GoLoco motif which mutated the important R to C. As stated above, the mutation of the C terminal R residue would abolish the binding activity, this might explain why paper by Luo et al. showed an increase of GDP release rate in the presence of the L7 protein.
Taken together, the GoLoco motif provides an interaction site for Go subunit and sen/es as GDI.
1.10 L7 mPNA Is localized in both cell body and dendrites In Purfcinje cells
Apart from the investigations of the interaction between the L7 protein and Go subunit, researchers also focused their study on the distribution of L7 mRNA in cerebellar Purkinje cells. Cerebellar Purkinje cells are bipolar neurons, its polarity
allows different mRNAs to be differentially distributed in different compartments.
The same is true for other neurons, like neurons in cortex and hippocampus.
Although most of the mRNAs synthesized in neurons are localized in cell bodies,
28 it Is now realized that more mRNAs are localized in dendrites than the originally thought (Eberwine et al., 2001; Miyashiro et al., 1994; Tian et al., 1999). These mRNAs include MAP2(microtubule-associated protein2) (Gamer et al., 1988),
Arc(activity-regulated cytoskeleton-associated protein) (Lyford et al., 1995),
CaMKIIa(Ca^^-calmodulin-dependent protein kinase lia subunit) (Burgin et al.,
1990), PEP19 (Sian et al., 1996), IP3 receptor (Furuichi et al., 1993), NMDA(N- methyl-D-aspartate) receptor (Gazzaley et al., 1997) and so on. Among these mRNAs, some of them are localized throughout the dendrites, like Arc and
CaMKIIa, others are in proximal dendrites, like MAP2. This finding has attracted investigators to study the mechanism that drives the differential distribution of mRNAs in neurons.
It has been shown that L7 mRNA is distributed uniformly in the axons, cell bodies and dendrites in cerebellar Purkinje cells (Bian et al., 1996). Wanner et al. also showed that L7 mRNA forms granules in both perikayon and in dendritic branches (Wanner et al., 1997). The quantitative analysis showed that the localization of the L7 mRNA in Purkinje cells is developmentally regulated. The concentration of L7 mRNA is decreasing from P9 to P25 in both cell bodies and dendrites in Purkinje cells. At P9 more L7 mRNA is localized in dendrites than at
P25. In primary cultures, L7 mRNA distribution in Purkinje cells showed a similar pattern as in vivo. In younger cultures, L7 mRNA signal detected in the dendrites is more than that in older cultures. In situ hybridization also revealed that L7
29 mRNA seemed to be concentrated at the branch points in the dendrites both in vivo and in cultures. Physiologically, when the primary cultures were treated with higher concentration of potassium which will depolarize the Purkinje cells, higher percentage of Purkinje cells showed dendritic localization of the L7 mRNA, and the level of L7 protein is dramatically increased. This increase of the L7 protein has been shown to be regulated at posttranscriptional level (Wanner et al.,
2000).These findings indicate that the distribution of the L7 mRNA in cerebellar
Purkinje cells is likely dependent on both the developmental cues and the stimuli received by Purkinje cells.
1.11 Cis-elements and trans-factors mediate mRNA translocation
Extensive studies on the dendritic localization of mRNAs have revealed the molecular mechanisms that localize them in dendrites. Both cis-elements and trans-factors have been shown to be involved in targeting these mRNAs to the dendrites. As in localized mRNAs in Drosophila and yeast, most dendritic localization signals have now been identified in the 3’UTR (3’untranslated region) with one exception of BC1 RNA whose localization signal resides at the S end of the RNA (Muslimov et al., 1997). The study of CaMKIIa subunit mRNA localization has provided the most detailed information about the cis-element that is involved in targeting the mRNA in dendrites.
30 1.11.1 Cis-element is required for mRNA translocation in neurons
In wild type animals, CaMKIIa subunit mRNA is localized to molecular layers of the hippocampus and lamina I of the frontal cortex which contain extensive dendritic arborizations and synaptic contacts. Mayford and colleagues first showed that a cis-acting signal in the 3’UTR is responsible for the dendritic
localization of CaMKIIa subunit mRNA (Mayford et al., 1996). By fusing the
CaMKIIa promoter to LacZ in the presence or absence of the 3’UTR of CaMKIIa,
two lines of transgenic animals were generated. Both of these two lines showed
a typical CaMKIIa expression pattern in the brain. But only the line with the
3'UTR showed dendritic localization of the transgene mRNA. This finding
suggests that the 3’UTR is important to localize the CaMKIIa mRNA in the
dendrites. Furthermore, the process of the transport of the mRNA from the cell
body to dendrites was monitored in a real time manner in hippocampal cell
culture (Rook et al., 2000). When the 3’UTR was dissected, two cis-elements
were identified to be responsible for the localization of the mRNA in dendrites
(Mori et al., 2000). One of the elements is a 30 nucleotide sequence which is
located at the 5'end of the 3’UTR. It is homologous to a sequence in the 3’UTR of
neurogranin whose mRNA is also located in dendrites in neurons. Another cis-
element was shown to have an inhibitory function on mRNA translocation in the
resting state, and this inhibition was abolished upon activation of the neuron.
Recently, Blichenberg and colleagues showed that the CaMKIIa 3’UTR may have
even more cis-elements involved in dendritic localization. These elements may
31 work cooperatively to regulate the transport of the CaMKIIa mRNA (Blichenberg et al., 2001). In another example, a dendritic locating signal has also been mapped to a 640bp sequence in the 3’UTR of MAP2 mRNA (Blichenberg et al.,
1999). All of these experiments cleariy demonstrate that the cis-element is indispensable, and without it the mRNA fails to be sorted into dendrites.
1.11.2 Protein factors are Involved in RNA localization in dendrites
In addition to cis-elements, RNA-binding proteins and cytoskeleton proteins have been shown to be involved in mRNA translocation. One of the well characterized proteins involved in RNA translocation in neurons is mammalian Staufen (Kiebler et al., 1999). Staufen is a double-stranded RNA binding protein, whose function in Drosophila has been well documented (Ferrandon et ai., 1997; Fuerstenberg et al., 1998; St Johnston, 1995). In rat hippocampal neurons, Staufen protein has been shown to be localized in the somatodendritic region and it colocalizes with mRNA-containing ribonucleoprotein particles in the distal dendrites. It is also evident that Staufen protein is associated with microtubules which are involved in the transport of certain mRNAs in neurons (Wickham et al., 1999). In vitro binding assays showed that Staufen protein can bind to several RNA transcripts
(Monshausen et al., 2001). These results suggest that Staufen could potentially mediate the movement of mRNA-containing RNP conr^lexes along the microtubules. Two proteins, MARTA1 and MARTA2(MAP2-RNA trans-acting proteins), have been shown to bind with high affinity to the 640bp cis-element in
32 the 3’UTR of MAP2 mRNA, and they do not show any significant binding activity to other dendritic mRNAs (Rehbein et a!., 2000). p-acfin mRNA is recently found to be localized in neurites. It is noticed that p-actin mRNA is present in granules together with components of translation machinery and its localization requires intact microtubules (Zhang et al., 1999a). Furthermore, an RNA binding protein
ZBP1 (zipcode binding protein 1) (Ross et al., 1997) has been show to interact with p-actin mRNA and colocalizes with P-actin-containing RNPs in cultured neurons. The colocalization of ZBP1 and microtubules was also detected in cultured neurons (Zhang et al., 2001a). Another RNA binding protein, TB-RBP
(testis brain RNA-binding protein), has been shown to interact with the Y box containing mRNAs ligatin and CaMKIIa. Disruption of the interaction between TB-
RBP and the Y box element causes mislocalization of CaMKIIa and ligatin mRNA containing RNPs. Also, kinesin has been indicated to play a role in the transport of these mRNAs. Taken together, these findings suggest that RNP particles may serve as units to move along cytoskeleton proteins and send certain mRNAs to their targeted positions in neurons. Upon reaching their destination, these RNP particles could associate with anchors to ftinction locally. These obsenrations raise an important question as to why certain mRNAs are localized in dendrites.
1.12 Local protein synthesis at synapse in dendrites
Different lines of evidence have shown that the localization of mRNA in dendrites plays an important role in neurons. The discovery of polyribosomes in the
33 postsynaptic spines supports the idea that protein can be synthesized locally in the dendrites(Steward and Levy, 1982; Steward and Reeves, 1988). This was further confirmed by the findings of other translation machinery components in dendrites(Gardiol et al., 1999; Tiedge and Brosius, 1996). More convincing evidence of translation in dendrites came from a study in which tagged mRNA constructs were transfected into dendrites and translated there(Crino and
Eberwine, 1996). Recently, Aakalu et al. showed that translation occurred in hot spots or areas close to synapses in dendrites usinga GFP reporter gene flanked by the 5'UTR(5’ untranslated region) and 3' UTR from CaMKIIa in hippocampal neurons(Aakalu et al., 2001). Protein synthesis in dendrites has also been shown to be regulated by neurotrophins(Aakalu et al., 2001; Kang and Schuman, 1996).
These observations cleariy showed that dendrites have the ability to synthesize new proteins, and these newly synthesized proteins can in tum change the composition of the cellular components locally in dendrites. Based on the findings of mRNAs localized in dendrites, the following proteins are very likely synthesized in dendrites; cytoskeletal proteins(MAP2, Arc), kinases(GaMKIIa), calcium binding proteins(dendrin, IP3r), G protein regulators(L7/pcp-2) and receptor proteins(glutamate receptors, glycine receptors). Several of these have been experimentally proven to be synthesized in dendrites(Scheetz et al., 2000).
These findings led researchers to investigate the mechanisms and functions of protein synthesis in dendrites. As a matter of fact, dendrites have a wide variety of receptors, ligand-gated ion channels and voltage-gated ion channels, and
34 many second-messengers play Important roles at synapses In dendrites. It Is very reasonable to expect that local protein synthesis would affect synaptic plasticity in response to the stimuli received by the dendrites. Synaptic plasticity reflects the ability of synapses to be modified In response to activation and to previous stimulation. The change In plasticity could be either structural or functional or both. Many studies have provided evidence to show that dendritic protein synthesis affects LTP(long term potentiation), LTF(long term facilitation) and LTD(long term depression).
The first evidence to show that LTP requires protein synthesis came from the usage of protein synthesis lnhlbltors(Otanl and Abraham, 1989; Stanton and
Sarvey, 1984). Frey et ai used anisomycin (ANI) to block protein synthesis and showed that LTP Is dependent on protein synthesis(Frey et al., 1988). This Is compatible with the finding by Kang and colleagues, who showed that BONF- and NT-3-induced long term potentiation requires a rapid protein synthesis In dendrites(Kang and Schuman, 1996). The dependence on dendritic protein synthesis of LTD was also observed In hippocampal neurons(Huber et al., 2000).
Huber et al showed that by using a cap analogue to compete with capped mRNA and hence to Inhibit translation and by using ANI to block protein synthesis, LTD failed to occur. Studies on LTF have also demonstrated the requirement of rapid protein synthesis In dendrites. Martin et al showed that 5-HT-induced LTF In
Aplysia sensory neurons required new protein synthesis at presynaptic side,
35
% : where protein synthesis increased 3 fold upon 5-HT application. Application of protein synthesis inhibitor blocked the LTF(Martin et al., 1997). To summarize, dendritic localized mRNAs make local protein synthesis in dendrites possible and newly synthesized proteins change the plasticity of dendrites and synapses in response to stimulation.
1.13 Work presented here
So far there is no experimental data to show that L7 protein is synthesized locally in dendrites and the molecular mechanism to direct L7 mRNA to dendrites is not clear. Here we present evidence to show that the L7 gene encodes two protein isoforms, L7A and L7B, that differ by 21 amino acids at their amino termini. The two forms are based on the selection of two alternative first exons. The result of this difference is that form L7B has two GoLoco motifs while L7A only has one.
Interestingly, both L7A- and L7B*encoding mRNAs are distributed in both the soma and dendrites, and this distribution is developmentally regulated. In an effort to examine whether the L7 3’UTR is involved in RNA localization in dendrites, modified forms of the RNA were expressed in Purkinje cells of transgenic mice and their subcellular distribution tested. In parallel, in vitro biochemical assays were used to define an RNA sequënce in the 3’UTR that is capable of fomiing an RNA-protein complex. Furthermore, FPLC column chromatography and sequence analysis by mass spectrometry have identified
36 two of these binding proteins, CGI-145 and UNR, CGI-145 has no known function yet, but UNR has been shown to be involved in translation and RNA stability.
37 lam
Figure 1.1 Schematic illuslratfon of the atnidure of Cerel)ellum(adapted and modified fm "Principfee of Neural Science" edited liy Eric R. Kandel, Jamee H. Schwartz and Thomas M. Jeeaell )
38 Biogenic amines Amino acids and Ions Noicidrenallnê. Olulamate. C e ? ' Uplds dopamine. ÛABA LFA RAF. pcoslaglandliis. leukdtienes. anandamlne. SIP 5-HT, hislnnHne acélylchûllne Psplldaa and proteins Angoiensln. Uadyklnln. liiiombin. braiS>esln, FSH. LH. TSH. endoiplilns ' Others Ligtil. odoianis. pheiMncoes. nucleotktes, opiates, caniiabinokte, endoiprtns
Ion ctianneis. Blolofltcal reaftonsea POKY. PLC-I». ProHteratlon. dffferentlaUon. adenylyl cyclases development, cell survival, anglogenesls. hypertroptty, cancer G-proteln4ndependenl effector molecules
GTP GTP GTP OTP Gene expression Adenylyl cyclases, PLCil. regulation mfilMllonofcAMP DAG, Increase In cAMP Bho production. Ce^. concentration ton channels. PKC phosptrodlesterases. Itanscriptton Nucleus phosphotlpases factors
Figure 1.2 An overview of trimeric G protein invoived pathways( Adapted from Trends in Phamacologlcal Science, 22, 368,2001) # • '
,
hRGMAlP bRET-RGS1 hRGSZI hRGSI
bRGS3 hRGS4
hRGS8 hRGSI 3 hRGSI 6 hRGSS
hRGS9 hRGS11 hGRSIO hRGSI 2 hRGSI 4
mConductin hplls hPDZ-RhoGEF mO-AKAP2 dLoco C2 cEGL-10 dPINS hRaplGAP mL7/pcp>2 8 mL7/pcp-2 A Figure 1.3 Comparison of RGS, AGS and other GoLoco motif containing proteins (Adapted and modified from TIBS vol. 24 Page 411 to 414,1999) 40 Chapter 2 MATERIAL AND METHODS 2.1 Mouse strains L7AAUG SV40 mice, L7 AAUG add-back mice and L7 AAUG add-back-PKC mice were generated and kept in an FVB/N background. L7GFP mice were originally generated in an FVB/N background and were crossed either with FVB/N or with B6C3F1. Mice in FVB/N background were also used for 5’RACE, in situ hybridization of L7 form A and form B, western blot and protein purification pilot experiments. 2.2 Rat strains Rats with a Sprague-Dawley Background were used for 3’RACE, in situ hybridization, western blot and immunohistochemistry. Adult rat cerebella used for protein purification were purchased from Pel-Freez./ 2.3 Human Cerebella Frozen human cerebella used in this study were obtained either from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, 41 Baltimore, Maryland, or from Harvard Brain Tissue Resource Center. All the tissue were obtained on dry ice and kept at 80"C(Table 2.1). 2.4 Constructs for transgenic mice 2.4.1 L7AAUG add-back construct Previously, in L7 AAUG vector, the L7 3’UTR was replaced by SV40 3'UTR and the resulting construct L7 AAUG SV40 was made(Feng Bian, 1998). In order to add polyA site mutated (U->G) L7 3’UTR back to the upstream of the SV40 3'UTR in the construct of L7 AAUG SV40, a PCR fragment was amplified using primers 3'UTR-U/G: ACTGACATGCATGCGCTAGTGCCAAGTGTTTTCTTGTTT T and Feng-8: GCTCCTTGAGCATGCTAGCCATCCT. The amplified fragment was then digested with SphI and inserted into the SphI site in the L7 AAUG SV40 construct and the resulting consctruct was L7AAUG add-back. 2.4.2 L7AAUG add-back-PKC construct The pseudo-substrate of PKC was inserted into BamHI site of the L7AAUG add- back construct and the resulting construct was L7AAUG add-back-PKC. The fragment of the pseudo-substrate of PKC was generated by annealing two DNA primers JD034: GATCCTTACACGTTCTTCTGCCTCAGGGCGCCCTTCCTGGC GAACCTCAT and JD035: GATCATGAGGTTCGCCAGGAAGGGCGCCCTGAG GCAGAAGAA CGTGTAAG. 42 2.5 Preparation of mouse tail DNA A piece of 1 -2mm mouse tail was digested at 55"C for several hours in 200 ul of digestion buffer which contains SOmM Tris(pH8.0), lOOmM EDTA, 0.5% SOS and lmg /ml proteinase K. After digestion, the mixture was phenol/chloroform extracted and ethanol precipitated. DNA pellet was then dissolved in lOOul of ddHaO, 1ul of the DNA is sufficient for PCR assay and 20ui of the DNA is sufficient for Southern blot. 2.6 Total RNA and mRNA isolation from mouse, rat or human tissue Total RNA was isolated from mouse, rat or postmortem human tissue using TRIzol reagent (Life Technologies, Inc.) following vendor's RNA isolation protocol. Human mRNA was isolated from the total RNA using PolyATtract® System(Promega) following vender’s protocol. Isolated mRNA was stored at - 80°C. 2.7 5'RACE of mouse and human L7 mRNA 2.7.1 S' RACE of mouse L7 mRNA Total RNA was isolated from PI4 FVB/N mouse cerebellum. Reverse transcription reaction was done using mouse L7 gene specific primer 3'UTR reverse wt which has a sequence of GCTAGTGCCAAGTG 111 lATTG. Primers used for the following PCR reactions are: Abridged Anchor primer(AAP, Life Technologies): GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG, L7 coding 43 Reversal: AGCTGGATCCGGGTGTTGACCAGCATATCCAT, L7 coding Reverse 2: AGCTGGATCCGCTTTCTGGGTTCTGGCCTGG. After reverse transcription, polyd(C) tall was added to the 5" end of the cONA by terminal deoxynucleotidyl transferase. The dC-tailed cONA was amplified by nested PCR using AAP, L7 coding Reverse 1 and AAP, L7 coding Reverse 2. PCR product was resolved on a 2% agarose gel and the desired band was excised and sequenced. 2.7.2 5'RACE of human L7 mRNA Total RNA was isolated from 1 year and 4 year old postmortem human cerebellum. Reverse transcription reaction was done using human L7 gene specific primer hL7-reverse 3 which has a sequence of ATCAG ii ii i GGGGGC CGAG. Primers used for the following PCR reactions are Abridged Anchor primer(AAP, Life Technologies), hL7-reverse1: TAATACGACTCACTATAGGGA GGTCAGGGGGCTTGTGTCGGGGGCTGG and hL7-reverse 4: ATCCATGCGG CGGCCCTGGG. After reverse transcription, polyd(C) tail was added to the S' end of the cDNA by terminal deoxynucleotidyl transferase. Then the dC-tailed cDNA was amplified by nested PCR using AAP, hL7 Reverse 1 and AAP, hL7- Reverse 4. PCR product was resolved on a 2% agarose gel and the desired band was excised and sequenced. 44 2.8 3'RACE of rat and human L7 mRNA 2.8.1 3'RACE of rat L7 mRNA Total RNA was isolated from adult rat cerebellum. Reverse transcription was done using adapter-d(T) primer which has a sequence of GACTCQAGTCQACAT CGAIII M 1 11111111111. Primers used for the following PCR reactions are JDO 23: GATGGAATGOAGAAAGG and adapter GACTCGAGTCGACATCG. After reverse transcription, the S' end of the cONA was amplified by PCR using JD023 and adapter. PCR product was gel-purified and sequenced. 2.8.2 3'RACE of human L7 mRNA Total RNA was isolated from 1 year and 4 year old postmortem human cerebella. Reverse transcription was done using adapter-d(T) primer which was used for the rat 3'RACE experiment. Primers used for the following PCR reactions are hL7-fon/vard 5: ACCAGGAGGGCTTCTTCAATC and adapter which was used for the rat 3'RACE. After reverse transcription, tfie S' end of the cDNA was amplified by PCR using hL7-fonvard 5 and adapter. PCR product was gel-purified and sequenced. 45 2.9 Making alpha-^P labeled DNA or RNA probes 2.9.1 Making alpha-^P labeled DNA probe for EGFP transgenic mice northern blot Plasmid DNA pGREENLANTERN^-1(Llfe technologies, Inc) was digested with Notl to release the GFP fragment and the GFP fragment was gel purified. GFP fragment was then labeled with alpha-“ P dCTP using Rediprime kit(Amersham) following vendor’s manual. After labeling reaction, unincorporated alpha-^P dCTP was removed by ethanol precipitation and the labeled probe was dissolved in TE(10mM Tris, ImM EDTA). 2.9.2 Making P-32 labeled DNA probe for human L7 northern blot PCR product amplified by using hL7-fonward 5 and adapter from 3'RACE experiment was used as template for making P-32 labeled probe. Alpha-^P dCTP was incorporated into DNA fragment by using Rediprime kit(Amersham) following vendor’s protocol. Lat)eled probe was then separated from unincorporated alpha-^P dCTP using Nucleotide Removal kit(Qiagen). 2.9.3 Making P-32 latwled RNA protxes for EMSA and UV cross-linking 2.9.3.1 Making full-length wt L7-3 UTR probe Plasmid DNA pGEM3Z-L7-3'UTR was linearized with Hindlll, the digestion mixture was then phenol/chloroform extracted to remove the enzyme, DNA was precipitated with ethanol and dissolved in TE(10mM Tris, Im M EDTA pHS.O). To 46 make sense L7 3'UTR probe, 1ug of linearized DNA was used as template and alpha-^P CTP was incorporated into the transcript by using T7 RNA polymerase. Labeled probe was then gel purified and stored at -80"C. 2.9.S.2 Making mutated L7-3 UTR probes In order to introduce mutations into the L7-3'UTR probes, a series of DNA oligos were synthesized as 3'primers and their sequences are as followings: 3'UTR Reversal: GCTAGTGCCAAGTGGTTTTCAGGGGCC: 3'UTR Reverse 2: GTGC CAAGTGGTTTTC; 3'UTR Reverse 3: CAAGTGGTTTTCAGGG; 3'UTR Reverse 4: TGGTTTTCAGGGGCCA; 3'UTR Reverse 5: GCTAGTGCCAAGTGGCAGGG GCC; 3‘UTR Reverse 6: GCTAGTGCCAAGTGTTTTCTTGTTTTCAGGG; 3'UTR Reverse 7: GCTAGTGAGCTGTGGTGTGCAGGG; 3'UTR Reverse 8: GCTAGTG AGCTGTGTTTTATTGTGTGCAGGG: 3'UTR Reverse 9: GCTAGTGCCAAGTGG GGGGGGGTTTTCAGGG; 3'UTR Reverse 10: GCTAGTGCCAAGTGTTTTATTG CAGGGG: 3'UTR Reverse 11: TGTTTTATTGTTTTCAGGGG. pGEM3Z-L7- 3'UTR was used as template and T7 primer was used as 5'primer. PCR products were purified either by agarose gel or by PCR purification kit(Qiagen). Purified PCR fragments were then used as template and alpha-^P CTP was incorporated into the transcript by using T7 RNA polymerase. Labeled probes were then gel- # purified and stored at -80“C. 47 2.9.3 3 Making RNA ollgo probes 5 RNA oligos were synthesized according to the sequence of L7 3'UTR. Their sequences are: oligol: UGGGCCUCCCACUGGCCCCUGAAAACAAUAAA; ollgo 2: UGAGAGUUCUAGCCAUCCUGGGCCUCCCAC; oligo 3; CUGAAAACA AUAAAACACUUGGCACUAGC; oligo 4: CACUUGGCACUAGC and oligo 5: CUGAAAACAAUAAAA RNA oligos were end-labeled with Gamma-^P ATP by 14 kinase. End-labeled probes were then purified by 10% polyacrylamide and stored at -80“C. 2 . 1 0 Northern blot Certain amount of total RNA or mRNA was dried down by Speed Vac. The RNA was resuspended in 10 ul of Northern loading buffer which contains 50% of formamide, 2.2M of formaldehyde, O.Sx formaldehyde gel-running buffer, 5% glycerol, 0.1 mM EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol FF. Dissolved RNA was then heated at 65*’C for 15 min and chilled on ice before it was loaded onto a 1% formaldehyde gel. The gel was run at 5v/cm for 3-4 hours until the leading edge of the bromophenol blue is 1 - 2 cm away from the edge of the gel. The gel was then washed with 5 changes of ddHgO for 20 mins each to remove the formaldehyde from the gel. Following the washing, the gel was stained with 0.5ug/ml Ethidium Bromide in a 50mM NH 4AC buffer for 15 mins, the gel was then destained with ddH20 for 1-2 hours until the background was completely removed. The gel was then photographed with a mler beside the gel 48 using a UV fluorescent gel documentation system(BioRad). After the picture being taken, the gel was placed in 10XSSC for 0.5-1 hour. Then the RNA was transferred from the gel to a nylon membrane using 20xSSC on a gel transfer apparatus for 16-22 hours. After the transfer, the membrane was rinsed with ddH20 to remove agarose and salts. Then the merribrane was baked at 80*C in a vacuum oven for 1 - 2 hours. Baked membrane was pre-hybridized for 2 hours at 65°C in a pre-hybridization solution which contains SxOenhardts, 4xSET, 0 . 1 % SDS, 1 xphosphate/pyrophosphate buffer, 5ug/ml of salmon sperm DNA and 1 Dug/ml of yeast tRNA. Then ^P-labeled denatured probe was added to the hybridization buffer and allow it to hybridize to the target mRNA for 16-22 hours at the same temperature. After hybridization, the membrane was first rinsed in 2XSSC to remove residual hybridization buffer, then the membrane was washed in a sequential buffers of 2XSSC, 1 XSSC, 0.5XSSC, 0.1XSSC until desired signal was detected while keeping the background low. Autoradiography was then performed to visualize the signal either by exposure to a Kodak X-ray film or by exposure to a Phospholmager screen. The Phospholmager screen was scanned on a Phospholmager driven by lmageQuant(Molecular Dynamics). 2.11 Electrophoretic mobility shift assay(EMSA) In EMSAs 1.0X1 O'* cpm of ^P-labeled RNA probe was incubated for 15 minutes with various amount of protein sample on ice in the presence of lOug heparin, 200ng yeast tRNA in a buffer contaninglOmM HEPES(pH8.0), 3mM MgCk, 49 40mM KCI, 5% glycerol and 1mM OTT. In competition experiments, unlabeled probe was mixed with labeled probe either before or after the addition of protein sample. The protein bound RNA was resolved from free RNA probe by a 5% native polyacrylamide gel. Gels were dried and bands were visualized by autoradiography at -80“C. 2.12 UV cross-linking of RNA-protein complex Binding reactions were carried out on ice with IXIO^cpm “ P-labeled RNA probe and various amount of protein sample in a buffer contaning lOug of heparin, 2 0 0 ng of yeast tRNA, lOmM HEPES(pHB.O), 3mM MgCI2, 40mM KCI, 5% glycerol and 1 mM OTT. After 10 minutes, the samples were irradiated by UV light for 150 seconds using a UV-crosslinker(Rsher Scientific, 120,000uJ/cm^). After irradiation, the samples were digested with lOOug/ml RNase A at 3 7*0 for 30 minutes( for Gamma-“ P ATP end-labeled RNA oligo, this step was skipped). The UV cross-linked products were resolved on a 10% SDS-PAGE. Gels were dried and the bands were visualized by autoradiography at -80**C. 2.13 Making S-35 labeled probes for in situ hybidization 2.13.1 Making L7 antisense probe DNA plasmid L7H0M1 which contains partial sequence of L7 cDNA was linearized with Hindlll, the digestion mixture w§& then phenol/chloroform extracted to remove the enzyme, DNA was precipitated with ethanol and 50 dissolved in TE(10mM Tris, ImM EDTA pH8.0). To make antisense L7 probe, lug of linearized DNA was used as template and alpha-^S UTP was incorporated into transcript by T7 RNA polymerase. 2.13.2 Making SV40 3'UTR antisense probe DNA plasmid pGEM3Z-SV40 3'UTR which contains SV40 3'UTR was linearized with Hindlll, the digestion mixture was then phenol/chloroform extracted to remove the enzyme, DNA was precipitated with ethanol and dissolved in TE(10mM Tris, ImM EDTA pHS.O). To make antisense SV40 3'UTR probe, lug of linearized DNA was used as template and alpha-^S UTP was incorporated into transcript by T7 RNA polymerase. 2.13.3 Making GFP antisense probe DNA plasmid pBS-GL which contains partial sequence of GreenLantem(Life Technologies, Inc.) was linearized with Xhol, the digestion mixture was then phenol/chloroform extracted to remove the enzyme, DNA was precipitated with ethanol and dissolved in TE(10mM Tris, ImM EDTA pHS.O). To make QL antisense probe, lug of linearized DNA was used as template and alpha-^S UTP was incorporated by T3 RNA polymerase. 51 2.13.4 Making human L7 antisanaa probe Human L7 cDNA fragment was obtained by RT PCR. Primers used for the RT- PGR are: hL7*forward 5: ACCAGGAGGGCTTCTTCAATC; hL7-reverse liTAATACGACTCACTATAGGGAGGTCAGGGGGCTTGTGTCGGGGGCTGG. Gel-purified RT-PCR product was used as template for in vitro transcription. Alpha-^^S UTP was incorporated into the transcript using T7 RNA polymerase. 2.13.5 Making human CaBP antisense probe Plasmid containing human CaBP cDNA fragment was linearized by BamHI, digestion mixture was phenol/chiorform extracted and ethanol precipitated. DNA pellet was dissolved in TE(10mM Tris, ImM EDTA). To make antisense probe, lug of linearized DNA was used as template and alpha- UTP was incorporated into the transcript using T7 RNA polymerase. 2.13.6 Making mouse exon 1A and IB specific antisense probes Two DNA fragments corresponding to the two exon 1's for mouse L7 form A and form B were amplified by PCR. Exon 1 A was amplified using primers L7-forward 3: GOG CGAATTCGATTCTTAGTACTGTCÔGCCÀ and L7 5’UTR Reverse 2: TAATACGA CTCACTATAGGGAGGCAGGGAAATGGGGCTCAGAAG. Exon IB was amplified using primers L7-forward 2 : GGGCAGGTCAGGGAAGGAGAC and L7 5’UTR reversel : TAATACGACTCACTATAGCCATAGTCCTCACGGGTC TGCAGAATTTCT. Amplified fragments were gel purified and sequenced. To 52 make antisense probes, 1 ug of each PCR fragment was used as template, alpha-^^S DTP was incorporated into transcript by T7 RNA polymerase. 2.13.7 Making rat L7 antisenae probe Rat L7 cDNA 3'end amplified by JD023 and adaptor in the 3’RACE experiment was reamplified using primers JD023 and rat reverse-!? : TAATACGACTCACT ATAGGGAGGGCTAGTGCCAAGTAF111ATTAC. PCR product was gel purified and sequenced. To make antisense probe, lug of this PCR fragment was used as template, alpha-^S DTP was incorporated into RNA transcript by 17 RNA polymerase. 2.13.8 Making rat CGI-145 and UNR antisense probes Total RNA was isolated from adult rat cerebellum. Rat CGI-145 and UNR cDNA fragments were amplified by RT-PCR using mouse primer pairs mCGI forward2: GGGAGACTGTGCCAGTTT, mCGI Reverse2: TAATACGACTCACTATAGG GCGCATTTACCAAATCTTCTGGCGAGAGC and UNR FI CCACGTCCACAAA GCACATCATTAA and UNR R 1 TAATACGACTCACTATAGGGAGGCATAGGG CCAACAAAGTAACAGTATG. PCR products were gel purified and sequenced and were used as template for in vitro transcription. To make antisense probe, alpha-^S UTP was incorporated into the transcript by T7 RNA pdoymerase. 53 After in vitro transcription reaction, the mixture was phenol/chloroform extracted followed by chloroform extraction. The labeled probe was ethanol precipitated in the presence of 20ug of yeast tRNA(Life Biotechnologies, Inc) and 2.5M ammonium acetate. Pellet was washed with 75% ethanol and dissolved in DEPC-treated water. Precipitate the probe again with ethanol in the presence of ammonium acetate to remove most of the unincorporated alpha-^S UTP. The probe was then washed with 75% ethanol and dissolved in 30ul of DEPC-treated water. 2.13.9 Quantitation of radioactivity of aipha-^ UTP labeled probe One microliter of S-35 labeled probe was precipitated with 5% trichloric acid(TCA) in the presence of 500ng of BSA on ice for 10 minutes. The precipitates was filtered with glass fiber filter paper(Fisher Scientific)using a vacuum pump and the filter was then washed twice with 5% TCA and dried in a vacuum oven. The radioactivity of the filter paper was measured using a scintilation machine. After calculation, the probe was diluted in hybridization buffer (Amresco) to a final concentration of 1X10^cpm/50ul and was kept at - 80°C. 2.14 In situ hybridization Mouse or rat brain was quickly dissected out after the animal was sacrificed. Fresh mouse or rat tissue was then left immediately on dry ice to freeze it, and 54 the frozen tissue was either sectioned right away or stored at -SO^C. Human cerebeila were obtained on dry ice from tissue bank and were stored at -SOX. For in situ hybridization, all the tissues were sectioned on a cryostat at a thickness of 12um and were stored at 80"C. Immediately before the experiment, the sections were taken out of -80°C freezer and were left on a 37"C heating plate for 15 minutes. The sections were first fixed with 4% paraformaldehyde, lXPBS(pH7.2) for 10 minutes followed by two changes of in 1XPBS to remove paraformaldehyde, then the sections were incubated with 0 . 1 M Triethanolamine(TEA)(pH8.0), 0.25% acetic anhydride for 10 minutes. After the TEA treatment, the sections were transferred to 70% ethanol for 1 minute followed by 80% ethanol for 1 minute, 95% ethanol for 2 minutes and 100% ethanol for 2minutes. To permiablize the cell membranes of the tissue, the sections were treated with 100% chloroform for 5 minutes followed by the incubation with 100% ethanol for 1 minute and 95% ethanol for 1 minute. After the pretreatment, the sections were air-dried for 2 0 minutes at room temperature. While the sections were being dried, S-35 labeled probe(s) was diluted in hybridization buffer(Ameresco) to 1X10^cpm/50 ul and DTT was added to a final concentration of 0.1 M. The probe(s) was denatured at 90"C for 3 minutes to unfold the secondary structure and was left on ice for use. 50ul of denatured probe was added onto each slide and the slide was covered with cover glass. After the probe was uniformly spread out between the slide and the cover glass, the cover glass was sealed with Qurr(BDH Laboratory Supplies)and the probe 55 was allowed to hybridize to the target at 55"C overnight. After the hybridization, the sealing was peeled off and the slides were left in 2XPBS to get rid off the cover glass. After the removal of cover glass, the sections were washed twice with 2XPBS, 5mM DTT at 45 degree for 15 minutes each. In order to remove the excess amount of free probe and the non-specifically hybridized probe, the sections were incubated with 20ug/ml RNase A at 37"C for 30 minutes. Following the RNase A treatment, sections were washed with 5 changes of 2XPBS at 37*C for 15 minutes each to completely remove RNase A. Then the sections were washed with 2 changes of 0.5XPBS at 37*C for 15 minutes each. Depending on the composition and the length of the ribo-probe used for hybridization, 4 changes of 0.1XPBS can be used to wash the sections at a higher temperature ranging from 65 to 8 5 X . After the final wash, the sections were dehydrated with a sequential treatment of ethanol, 1 minute in 0.3M NaAc, 75% ethanol, 1 minute in 0.3M NaAc, 80% ethanol, 1 minute in 95% ethanohand 5 minutes in 100% ethanol. After dehydration, slides were air-dried arid exposed to X-ray film overnight at room temperature. Signals on the X-ray film were checked to determine whether the final wash condition was optimal or any changes were necessary. Sections which give good signal were dipped with Emulsion- 2(Eastman kodak). Dipped sections were kept at 4 X for a suitable time period depending on the signal of the sections. Then the sections were developed with developer D-19 and fixed with fixer. Developed sections were counter-stained with cresyl-violet and mounted using permount. The mounted sections were 56 viewed under the microscope and both dark field and bright field images were taken. 2.15 Western blot Appropriate amount of pure protein or protein extract was separated on a SDS- polyacrylamide gel according to Laemmli. Then the protein in the gel was transferred to a nitrocellulose membrane in a transfer buffer containing 25mM Tris, 192mM Glycine and 20% methanol. After the transfer, the efficiency of the transfer was checked by staining the blot with 0.2% Ponceau S for 2 min. The membrane was then blocked for 1 hour with blocking buffer containing SOmM Tris-HCI, pH7.5, SOOmM NaCI, 1% BSA and 5% Dry milk. After the blocking step, the membrane was washed twice for 5 minutes each with TEST buffer containing 50mM Tris-HCI, SOOmM NaCI and 0.05% Tween-20. Primary antibody diluted in 1% BSA/TBST was used to incubate the membrane for 1 hour at room temperature. The membrane was then washed with TEST once for 15 minutes and four times for 5 minutes each. HRP-labeled secondary antibody diluted in 1% BSA/TBST was incubated with the -membrane for 1 hour at room temperature, and the membrane was then washed with TEST once for 15 minutes and 4 times for 5 minutes each. The visualization reaction was done by using Westem blot Chemiluminescence Reagent kit(NEN) and the signal was visualized by X-ray film. Mouse L7 polyclonal antibody E2 and PEP1 were diluted 1,000 fold, mouse CaEP monoclonal antibody(Sigma) was diluted 5,000 fold, 57 GFP monoclonal antlbocly(Clontech) was diluted 1,000 fold, GFP polyclonal antibody(Clontech) was diluted 200 fold, HRP-labeled secondary antibody was diluted 2,000 fold. 2.16 Immunohistochemlstry with fluorescent secondary antibodies Floating sections were prepared by cutting the tissue in 40 microns and stored in 1XPBS in cold room. The floating sections were first blocked for 30 min at room temperature with 2% donkey serum in PBST which contains 1XPBS and 0.3% Triton X-100. After the withdrawal of the donkey serum, primary antibody diluted in PBST was added and incubated with the sections for 1 hour at room temperature. Sections were then washed 3 times with 1 XPBS for 5 minutes each. Following the washing, the sections were incubated with Cy2, Cy3 or Alexa 488 labeled secondary antibody in PBST for 1 hour at room temperature. The sections were washed 3 times with 1 XPBS and once with ddH20 for 5 minutes each. Each section was transferred to a slide and mounted with Vectorshield(Vector). The images were taken under confocal microscope(Bio- Rad) using LaserSharp program. Mouse L7 polyclonal antibody B2 and PEP1 were diluted 1,000 fold, mouse CaBP monoclonal antibody was diluted 10,000 fold, Cy2 labeled secondary antibody was diluted 200 fold, Cy3 labeled secondary antibody was diluted 800 fold, Alexa 488 labeled secondary antibody was diluted 1,000 fold. 58 2.17 Preparation of protein extract from wt or GFP transgenic animats One cerebellum or similar sized brain cortex was homogenized in 1 ml of homogenization buffer containing 0.5XPBS, 2mM MgCI2, 0.02% NP-40 and 0.1 mg/ml deoxycholate. The homogenate was centrifuged at 14000rpm for 15 minutes in cold room. Transfer the upper phase to a tube and determine the protein concentration by Bradford method using BOA kit(Pierce). 2 . 1 8 Fluorescence measurement of GFP protein by ftuorometer To determine the intensity and the wavelength of emission of the GFP protein expressed In mouse cerebellum, protein extracts from wild-type cerebellum and from the brain cortex as well as cerebellum of the GFP transgenic mouse were used to measure their emission spectra at a excitation wavelength of 484nm. Homogenization buffer which contains 0.5XPBS, 2mM MgCI2, 0.02% NP-40 and 0.1 mg/ml sodium deoxycholate was used as a control. The emission spectra were recorded from 495-580nm. 2.19 Measuring the fluorescence of GFP protein using fluorescence microscope Pure GFP protein(Clontech) and crude protein extracts from wild-type and GFP transgenic mouse cerebeila were loaded onto a nitrocellulose membrane using a dot blot apparatus, the fluorescence of the protein was visualized under a 59 fluorescence Axioscope 2 microscope. The images were taken using a spot camera and program. 2.20 Preparation of protein extract from mouse cerebellum Mouse cerebeila were homogenized in cold room in a homogenization buffer containing 1GmM HEPES(pH7.9), lOmM KCI, I.SmM MgCI2, O.SmM DTT, 0.5mM PMSF, 2ug/ul aprotinin, Im M EDTA, lug/m l leupeptin, 0.2mg/ml pefabtoc and 1ug/ml pepstatin. The homogenate was centrifuged at 2,000rpm for 15 minutes in cold room to remove nuclei and debris. After centrifugation, supernatant was transferred to an ultracentrifuge tube and 0 . 1 1 volume of high salt buffer, which contains 300mM HEPES, pH7.9, 1.4M KCI and 30mM MgCI2, was added. The supernatant was then centrifuged at 40,000rpm for Ihour at 4°C. After ultracentrifugation, the dear supernatant was removed and was dialyzed against a buffer containing 20mM HEPES(pH7.9), lOOmM KCI, 0.2mM EDTA, 20% glycerol, O.SmM DTT and O.SmM PMSF. After several changes of dialysis buffer, the protein extract was stored at -80"C. 2.21 Preparation of protein extract from ret cerebellum 20 adult rat cerebeila were homogenized in a homogenization buffer containinglOmM Tris-HCI, pH7.9, lOmM KCI, I.SmM MgCI2, OSmM DTT, O.SmM PMSF and 1/100 volume of protease inhibitor cocktail(Sigma). The fwmogenate was then centrifuged at 2,000rpm for 30min to remove nuclei and debris. After 60 centrifugation, supernatant was transferred to a new tube and was kept on ice, pellet was re-suspended in homogenization buffer and homogenized and centrifuged again . Two portions of the supernatant were combined and 0.11 volume of high salt buffer, which contains SOOmM Tris-HCI(pH7.9), 1.4M KCI, and SOmM MgCI2, was added. The supematant was then ultra-centrifuged at 40,000rpm for 1 hr at 4*C. After ultracentrifugation, the dear supematant was dialyzed against a dialysis buffer A containing 20mM Tris-HCI(pH7.9), 40mM KCI, 0.2mM EDTA, 10% glycerol, O.SmM DTT and O.SmM PMSF. After dialysis, the protein was stored at -80®C. 2.22 Purification of L7 3'UTR binding proteins from cerebellar protein extract DEAE-sepharose fast flow matrix was packed into a 30cm(L)x1.Scm(d) Econo column(Bio-Rad) with a bed volume of about 30ml. The column was equilibrated with buffer A containing 20mM Tris-HCI(pH7.9), 40mM KCI, O.SmM EDTA and 10% glycerol. Crude protein extract from 20 rat cerebeila was applied onto this column at a flow rate about O.Sml/min. After loading the sample, the column was washed with 2 bed volumes of buffer A. Following the washing, protein bound to the column was eluted with 3 bed volumes of gradient from 40 to 400mM KCI. Protein binding activity was eluted from ISOmM to 210mM. The pooled fractions were dialyzed against 1 liter of dialysis buffer A for 4 hours and clarified by centrifugation for 30 minutes at 2S,000 rpm using a Beckman VTi28 rotor. 61 A 5 ml HiTrap Heparin-sepharose high performance column(Amersham Pharmacia Biotech) was equilibrated with buffer A before the DEAE-sepharose pool was applied to it. The column was then washed with 3 bed volumes of buffer A. Bound proteins were eluted with 5 bed volumes of gradient from 40 to 700 mM KCI. Protein binding activity was eluted from 450 to 530 mM KCI. The pooled fractions were dialyzed against 1 liter of dialysis buffer 8 containing 2 0 mM Tris-HCI(PH7.9), 1M (NH 4)2S0 4 . 0.2 mM EDTA, 10% glycerol, 0.5mM DTT and O.SmM PMSF. Dialyzed fractions were clarified by centrifugation for 30 minutes at 15,000 rpm in a Son/al rotor. A 5ml HiTrap Phenyl-sepharose high performance column(Amersham Pharmacia Biotech) was equilibrated with buffer B containing 20mM Tris-HCI(pH7.9), 1M (NH4)2S 0 4 , O.SmM EDTA and 10 % glycerol. Supematant from the centrifugation of the dialyzed heparin peak fractions was then applied onto this column. The column was washed with 2 bed volumes of buffer B. Bound proteins were then eluted with 5 bed volumes of gradient from 1M to OM (NH 4)2S0 4 . Protein binding activity was eluted from 120 to 0 mM (NH 4)2S0 4 . For both the SOKD and 95KD proteins, pooled fractions were dialyzed against buffer buffer A. Dialyzed fractions were clarified by centrifugation for 30 minutes at 15,000 rpm in a Sonral rotor. 62 Two tandem connected 1ml SP-sepharose HiTrap coiumns(Amersham Pharmacia Biotech) were equilibrated with buffer A. Peak binding activity fractions for the 50KD protein were loaded onto this column. The column was washed with 10 bed volumes of buffer A. Bound proteins were then eluted with 10 bed volumes of gradient from 40mM to 1 M KCI. Protein binding activity was eluted from 740mM to SOOmM. Pooled fractions were dialyzed against buffer A and clarified by centrifugation for 30 minutes at IS.OOOrpm in a Son/al rotor. A 1ml Mono Q HR column(Pharmacia Amersham Biotech) was equilibrated with buffer A, peak binding activity fractions of the 50KD protein was loaded onto this column. The column was washed with 20 bed volumes of buffer A, and the bound proteins were eluted with 20 bed volumes of gradient from 40mM to 1M KCI. Protein binding activity was eluted from SSOmM to 600mM. After the binding activity assay was done, a 50KO band was resolved on an SDS-PAGE and excised. After Mono Q HR column was equilibrated with buffer A, pooled peak activity fractions for the 95KD protein from HIC column was loaded onto this column. The column was washed with 15 bed volumes of buffer A and bound proteins were eluted with 20 bed volumes of gradient from 40mM to 1 M KCI. Protein binding activity was eluted from 360mM to 460mM. Protein binding activity was analyzed and a 90KD band was resolved on SDS-PAGE and excised. 63 2.23 Concentrate protein samples by TCA precipitation 100% TCA solution was added to protein samples to make the final concentration of TCA 10%. The sample was left on ice for 1 hour and then was centrifuged at 14,0G0rpm for 15 mins in cold room. Pellet was washed with pre chilled 100% acetone and then dissolved in 40mM Tris, pH8. 2.24 SDS-PAGE of fractions and gel staining Appropriate amount of fractions from DEAE-sepharose and Heparin-sepharose were mixed with 5X loading buffer and heated at 95 degree for 5 minutes, then the samples were separated on a 10% SDS-polyacrylamide gel according to Laemmli. The gels were stained with Coomassie Brilliant blue R250. Certain amount of fractions from Phenyl-sepharose, SP-sepharose and Mono Q were separated on a 10% SDS-PAGE. The gels were silver-stained with Silver Stain Plus kit(BioRad) 2.25 Sample preparation for in gel digestion and MALDi-MS Protein samples and different concentrations of standard BSA and IgG were resolved by a10% SOS polyacrylamide gel with a gel thickness of 0.5mm. The gel was stained with 0.1% Coomassie Brilliant blue R250 in 10% acetic acid, 50% methanol and 40% H20 for 30 minutes and destained for 3 hours. Protein 64 band was excised and was sent out on dry ice to Yale Cancer Center Mass Spectrometry Resource and The HHMI Biopolymer/W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. 65 Age years days Cause of death disorder 0 5 Congenital heart defect control 1 123 Dehydration control 4 258 drowning control 28 131 Congenital heart disease control 58 control 90 control Table 2.1 human cerebellum tissues used in the experiments 66 Chapters ANALYSIS OF THE S END OF L7 mRNA IN BOTH MOUSE AND HUMAN 3.1 5’ Differential expreasion of two forms of L7 mRNA detected by 5’ RACE The mouse L7/pcp-2 gene (henceforth called L7) has been reported to be specifically expressed in cerebellar Purkinje cells and in retinal bipolar neurons, and it has been shown to contain four exons which can be transcribed into a 465- nucleotide mRNA (PCD-5) which can in turn encode a 99 amino acid protein (Nordquist et al., 1988; Oberdick et al., 1988; Vandaele et al., 1991). It has been reported that the expression of L7 mRNA is developmentally regulated with both a higher expression level and relatively increased fractionation into dendrites in Purkinje cells of younger animals(Wanner et àl., 2000). As has been indicated(Feddersen and Beitz, 1999; Oberdick et al., 1988) L7 mRNA may have different 5'ends due to alternative splicing, and different forms of L7 mRNA may coexist in younger animals. To address this issue, 5’ RACE(Rapid amplification of cDNA Ends) was performed to amplify the alternative 5’ ends of L7 mRNA using total RNA isolated from postnatal day 14 cerebellum. An L7 gene-specific S' primer (S’ UTR reverse wt was) used for the reverse transcription gives rise to a full length L7 cDNA. After two runs of nested PCR, one major band and one minor band could be resolved on a 2% agarose gel (figure 3.1 A). Both of the two 67 bands were purified and sequenced. As shown In figure 3.1 A, band A corresponds to the previously known L7 clone PCD-5, but our sequence has an extra 30 nucleotides at its S' end (figure 3.1 B) and this makes the full length cDNA 495 nucleotides. W e named this sequence L7 form A. Band B in figure 3.1 A corresponds to a full length cDNA of 485 nucleotides which we named L7 form B. The difference between the sequences of form A and form B is that form B has an alternative exon 1 which was part of the intron 1 of the previously reported L7 gene (figure 3.1 B, see also figure 3.6). We named the exon 1 in form A exon 1A, and exon 1 in form B exon IB . Both form A and form B share the common exons 2 to 4. One remarkable difference between exon 1A and exon 1B is that exon 1A does not have any translation start codon while exon IB has one at the very 3’ end (figure 3.1 B). In form A, a translation start codon in the middle of exon 2 is used to initiate protein synthesis and this will give rise to a 99 amino acid protein as previously reported. In form B, the start codon in exon IB is used to initiate protein synthesis, and this start codon is in the same reading frame as the one in exon 2. This gives rise to an L7B protein that is identical to L7A except for the addition of 21 amino acids at its N-terminus. Thus form B encodes an L7B protein of 120 amino acids (see Figure 3.9). 68 3.2 L7 form A and form B mRNAs are detected both in cell body and dendrites in younger animals L7 mRNA has been reported to be localized both in the cell body and dendrites in cerebellar Purkinje cells. To examine whether this holds for both form A and form B, in situ hybridization was performed using exon 1A and exon IB specific ribo- probes. Because the L7 mRNA expression level and the distribution in Purkinje cells vary with age, 4 different aged (P7, PI 4, P21 and 6 month) wild-type mouse cerebeila were used for in situ hybridization (figure 3.2 and 3.3). As shown in figure 3.2, form A mRNA is expressed in all these different aged cerebellar Purkinje cells, with peak expression around the second to the third week after birth. One prominent property is that the localization of form A mRNA changes with age. In P7 and adult cerebeila, most of the mRNA is localized within the cell body, but in P14 and P21 cerebeila, a significant amount of the form A mRNA is visualized in the dendrites. As shown in figure 3.3, all of the different aged cerebeila tested express form B mRNA, with a peak expression around P I4. At P7 and P14 a significant amount of mRNA is detected in dendrites, but the amount of mRNA in dendrites decreases with increasing age. The major difference between form A and form B mRNA is that form B is dominant during the first two weeks after birth, but form A is dominant after P14(figure 3.2 and 3.3). This suggests that the expression and localization of from A and form B mRNA are developmentally regulated. 69 3.3 Characterization of the rat L7 gene In order to compare the mouse L7 gene to its close relative in rat and to prepare for the experiments described in chapter 5 in which the purification of L7 mRNA binding proteins from rat cerebellar extracts is described, the rat L7 gene was analyzed. As a first step, we did 3’RACE to amplify the 3'end of the cDNA, which covers the 3'UTR and part of the coding region. The sequence of this fragment showed very high homology to the mouse L7 3'end, and this fragment was used as probe for in situ hybridization(figure 3.4 A and B). The in situ hybridization data clearly showed that rat L7 mRNA is exclusively expressed in Purkinje cells in cerebellum, and the mRNA is localized both in the cell body and dendrites. Previously, we obtained the sequence of a rat chromosome fragment which covers the first several exons and introns of L7 gene. When we combined this chromosome sequence with the 3'end sequence amplified by RACE the full length cONA sequence for rat L7 was obtained, and this was confirmed by RT- PCR. When we compared this rat chromosome sequence to the mouse chromosome sequence(accession number S40022), we were able to identify the two putative A and B forms of rat L7. For b6th forms rat cDNAs are 94% homologus to their mouse counterparts, and rat proteins are 95% identical to their mouse homologues(figure 3.9). the rat L7 gene structure is exactly the same as in mouse (figure 3.6). To confirm the expression and distribution of rat L7 protein in Purkinje cells, both westem blot and immunohistochemlstry were performed. In figure 3.40, the rat L7 protein is distributed throughout the Purkinje 70 cells as in the mouse(figure 3.4 C), and In figure 3.10B, a protein of the correct size for L7 protein can also be detected by westem blotting using mouse L7 antibody. 3.4 Cloning of the human L7 gene Although the rodent L7 gene was characterized about 13 years ago, the existence, and especially the expression, of its human homologue has never been confirmed. To investigate the human L7 gene, we first did a TBLAST search in the Celera human database using the mouse L7 protein sequence, and a 455 nucleotide human genomic sequence fragment (GA_12188666) was fished out. Part of this sequence is homologous to mouse L7 exon 4. By using this sequence to perform BlastN search we were able to pull out human chromosome 19 fragments from both Celera (most updated, GA_x2HTBKPS824) and NCBI (most updated, NT_01 1145.4) human genome databases. After sequence analysis by the gene-finder program (powered by The Department of Cell Biology, BCM/The Sanger Center, UK), we were able to define four exons which can encode a protein that is highly homologous to mouse L7. Based on these sequences, DNA primers were synthesized. We first amplified a fragment of the 3’end of the last exon by RT-PCR usxing these primers. The initial in situ hybridization data using this fragment as probe clearly showed that this human mRNA is localized only in Purkinje cells as is mouse L7 mRNA. In order to confirm that this is the human L7 gene, we started to amplify the full length 71 cDNA. The first experiment we did Is 3’RACE and the 3’end of this gene was amplified and sequenced. Then a longer cDNA sequence was amplified which covers the 3’UTR, coding region and part of the 5'UTR. By comparing to the mouse L7 gene structure, we wanted to know whether its human homologue also has altemative 5’ends. To do this, 5’RACE was performed to amplify the 5’ends of the cDNA. A gene specific primer (hL7 reverse 3) in the 3’UTR was used for the reverse transcription to generate almost full length cONA. After two runs of nested PCR, two sharp bands were resolved on a 2% agarose gel (figure 3.5A). Both bands were purified and sequenced. As shown in figure 3.5A, band A corresponds to the 5'end of a 550bp. full length cONA and band 8 corresponds to the 5'end of a 492 bp. full length cDNA. As in mouse the difference of these two forms of cDNA in human lies in the usage of different exon 1’s. By comparing these human cDNAs and their encoded proteins to their mouse counterparts, the SSObp. cDNA corresponds to the mouse L7 form A, and it can also encode a 99 amino acid protein with an identity of 81% to.mouse L7 protein form A; the 492bp. cDNA corresponds to the mouse L7 form B, and it can encode a 120 amino acid protein with an identity of 84% to mouse L7 protein form B. Hence, we named the SSObp. cDNA human L7 form A, and the 492bp. cDNA human L7 form B. In mouse the L7 gene has two altemative exon 1’s, and each of them is spliced to the downstream exon 2. In the human L7 gene, exon 1B is spliced to the downstream exon 2 as in mouse and the DNA sequence of exon 1B is highly homologous to mouse exon IB . But human L7 form A has only 72 3 exons, since the 5’end of exon 1A is part of the intron 1 sequence in form B. Also, the 3’end of exon 1A is the exon 2 used by form B(figure 3.5B). The 5’end of human exon 1A is not homologous to mouse exon 1A. These data show that the mouse and human L7 genes have a conserved gene structure even though the human L7 exon 1A is different from mouse exon 1A. A summary of the L7 gene structure in rodent and in human is illustrated in figure 3.6, and their differential usage of exon 1 is compared. To confirm the expression of the human L7 gene and to confirm the size of its transcript, northern blotting was performed using cerebellar mRNA(figure 3.10A). As in mouse, L7 transcripts in human were shown as a broader band due to the two different sized transcripts, and the size range of the band is consistent with the one obtained by RT-PCR. 3.5 The human L7 mRNA Is found in both the Purkinje cell body and dendrites and the dendritic localization is developmentally regulated In order to compare the localization of human L7 mRNA in cerebellum to that in rodent, different aged human cerebeila were used for in situ hybridization. Like in mouse and rat, human L7 mRNA is specifically expressed in Purkinje cells in cerebellum(figure 3.7). In new bom (5 days) and 1 year old human cerebellum, 17 mRNA is found in both the Purkinje cell body and dendrites (figure 3.8). Developmentally, Purkinje cells in a 5 day old human are equivalent to those in a 1 to 2 week old mouse. And, in W , the distribution of L7 mRNA in 5 day and 1 year old human is similar to what we saw in P7 and P I4 mouse. In 4 year old 73 human cerebellum, most of the L7 mRNA is localized in the Purkinje cell body and proximal dendrites with little signal detectable in the distal dendrites. For human ages 14(data not shown), 28 and 58 years, L7 mRNA can be detected mainly in the cell body and proximal dendrites (figure 3.8). In 90 year old cerebellum, the abundance of L7 mRNA is significantly reduced, and the mRNA can only be detected in the cell body(figure 3.8). 3.6 Human L7 protein has two GoLoco domains as in rodent but has more potential modification sites than in rodent The rodent and human L7 protein sequences were compared. One prominent feature of the amino acid composition of these proteins is the high Ser and Thr content(Table 3.1). This feature is even more prominent in human than in rodent. In rodent, Ser and Thr together account for 11 *13% of the total amino acids in both forms A and B; in human, Ser and Thr together account for 20% of total amino acids in form A and 18% in form B. The percentage of Glu and Asp residues is another feature that differs between rodent and human. On the other hand, the content of Gly and Pro, two destablizers of protein secondary structure, is consistently high between rodent and human. When posttransiational modification sites were analyzed(ExPASy, proteomics tools), human L7 proteins showed more potential modification sites due to the difference of the amino acid composition. As shown in table 3.1 and figure 3.9, the increase of Ser, Thr and the decrease of Glu in the human L7 protein allows it to have more potential N- 74 myristoylation and phospholation sites. Another consen/ed property between rodent and human L7 proteins is the GoLoco domain. For both rodent and human, form A protein contains only one complete GoLoco domain, while form B protein contains two complete GoLoco domains. This suggests a functional difference between form A and form B proteins. 3.7 Human L7 protein is specifically expressed in Purkinje cells To confirm the expression of the human L7 protein, both westem blotting and immunohistochemical staining were performed. On the westem blot, human L7 protein can be detected using anti-mouse L7 antibody. The expression level of L7 protein changes with age. At 28 years of age, an expression peak was detected. It is possible that the two bands detected on the blot correspond to form A and form B(figure 3.1 OB). As expected, human L7 protein is distributed throughout the Purkinje cells and this also suggests that it would have a similar «• function as its rodent homologues(figure 3.10 C ).' 75 Figure 3.1 5’RACE of mouse L7 cDNA and mouse L7 genomic sequence from exon 1A to exon 2 A. Agarose gel image showing two amplified. PCR products corresponding to the 5'ends of L7 cDNAs. Band A corresponds to the 5’end of form A and band B corresponds to the 5'end of form B. B. Mouse genomic sequence showing the region from exon 1A to exon 2. Underlined regions are exon 1A, exon IB and the 5'end of exon 2. Two horizontal arrows indicate the two altemative transcription start points for exon 1A and IB . The vertical arrows indicate the splicing sites, ® and v|) are the 5' splicing sites, and the 3’splidng site is indicated as 3’ss. The translation start codon for form A is indicated by short arrows, and the start codon for form B is indicated by asterisks. 76 B AATGAATGGATTCTTAGTACTGTCCCCCAAGAGATAGTAGGTACTAGGATTTAGGGGCAC # » ^ exon lA t s p 9 TTCTGAGCCCCATTTCCCTGGTAAGTGTCCCAACCCCCCAAATCAACCCAAGCCTGGTCT mouse CAATCTAGGACAGTGGTAGAATGCTGTCCCTAGAGTCAGTACCATGTGAAATTGTGCTGC AGGCAGGGGCCCCAGGCTGGGAGGTGGGGGTTGGGGGAGTCAGGGCAGGTCAGGGAAGGA t s p GACTCAGGTTTCATTTAGAGAAATTCTGCAGACCCGTGAGGACTATG&TGAGAGCAGAGA exon IB *** M TGGGAAGGCAGGCACTGTTTCGGGTGGATGCTGTCTGGAAGACAGGGAAGGCACAGACCA 3 ’SS AACTAAACCAATCACGTCTGTCCCCAAOGCAGGTTCACCGGACCAGGAAGGCTTCTTCAA , AGSPDQEGFFN CCTGCTGACCCACGTGCAGGffCG^TCGGATGGAGGAGCAG e x o n 2 LLTHLQGORMEEQ Figure 3.1 5’RACE off mouse L7 cDNA and L7 genomic sequence ffrom exon 1A to exon 2 Figure 3.2 In situ hybridization of mouse L7 form A mRNA at different ages In situ hybridization of mouse L7 form A mRNA Is shown at ages P7, P I4, P21 and adult(6 months). The left panels are the bright field images and the right panels are the corresponding dark field images. Arrows indicate different layers within the cerebellum. EGL: external granui cell layer, M: molecular layer, P; Purkinje cell layer and G: granui cell layer. The scale bar shown is 200 um. 78 Figure 3.2 in situ hybfkN» Non off mouse L7 fbnn A mRNA si différent ages 79 Figure 3.3 In situ hybridization of mouse L7 form B mRNA at different ages in situ hybridization of mouse L7 form 8 mRNA is shown at ages P7, P14, P21 and aduit(6 months). The left panels are the bright field images and the right panels are the corresponding dark field images. Arrows indicate different layers within the cerebellum. EGL; external granui cell layer, M: molecular layer, P: Purkinje cell layer and G: granui cell layer. The scale bar shown is 200 um. 80 Figura 3.3 in situ hybridialion ofm oiiM L7fonn B mRNAat différant âges 81 Figure 3.4 In situ hybridization of rat L7 mRNA and immunohistochemical staining of rat L7 protein A. Bright field of in situ hybridization. B Dark field of in situ hybridization. Arrows in A and B indicates Purikinje ceil layer(P), molecular layer(M) and granui cell layer(G). C. immunohistochemistry staining of mouse L7 protein by CyS, Purkinje cell body and dendrites are indicated. D. Immunohistochemistry staining of rat L7 protein by Cy2, Purkinje cell body and dendrites are indicated. 82 iSSSiRf °urk njL‘ cell some Purkinje cell su'ti.i It; r Or I tes dendrites Figure 3.4 in situ hybddbsdon of rat L7 mRNA and immuno- histochomicai staining of rat L7 pretain 83 Figure 3.5 5’RACE of human L7 cONA and human L7 genomic sequence from exon 1B to exon 2 A. Agarose gel image showing two amplified PCR products corresponding to the 5'ends of L7 cDNAs. Band A corresponds to the S end of form A and band B corresponds to the 5’end of form B. B. Human genomic sequence showing the region from exon 18 to exon 2. Underlined regions are exon 1 A, exon 1B and the 5'end of exon 2. Two horizontal arrows indicate the two altemative transcription start points for exon 1A and IB. The vertical arrows indicate the splicing sites, 5’ss is the 5’ splicing site, and the 3’ss is the 3’splicing site. The translation start codon for form A is indicated by short arrows, and the start codon for form B is indicated by asterisks. 84 B CCGGGATT^GAAGGAGACTCATGTCTCATTCAGAACAGCTCTGCAGAGAGGATCGGCGQ human tsp e x o n IB 5'ss GGACCATGQTG^GAGCTGAGGAAGTGGGGACGGCAGGCCCCGGGGTCAGGGTGTGAGCAA *** (gp * exon lA A M B GCTGGCTAQGAAGTTTGGGGAGGGGCCCCCAAGCTGAAGGGCCAACCAGACTGAGCGAGT 3'ss I S CTGTCCCCAOGCGGGCTCCCCAGACCAGGAGGGCTTCTTCAATCTGCTGAGCCACGTGCA agspdqegffnllshlq GGGCGACCGGATGGAGGGACA<3CGCTGTTCA e x o n 2 GDRMEGQRCS Figure 3.5 5’RACE of human L7 cDNA and human L7 genomic aaquanca from axon IB to axon 2 Figure 3.6 Comparison of human and rodent L7 gene structure Mouse(mL7), rat(rL7) and human L7(hL7) gene structures are compared by aligning their exons and introns. Open and filled boxes are exons and the horizontal lines between the boxes are in trons. Rodent exon 1A is indicated in the legend, it doesn’t share homology with human exon 1A. Human and rodent exon 1B is indicated by the same box because of their high homology. The 5’end of human exon 1A is indicated by a hatched box. Open boxes indicate downstream exons. Conserved exons 2,3 and 4 are indicated by Ex2, Ex3 and Ex4. Dotted lines are used to indicate the process of splicing. 86 a t g a t g TGA mL7 rL7 hL7 □ Exon 1A rodMitexonIA human and rodent axon IB S end o f human exon 1A downatraam axona Figure 3.6 Comparison of human and rodent L7 gene structure Figure 3.7 In situ hybridization of mouse, rat and human L7 mRNAs Comparison of human L7 mRNA localization to its rodent homologue in cerebellum. The magnification of each image is indicated in each panel. M; molecular layer, P: Purkinje cell layer, G: granui cell layer. 88 Figura 3.7 in situ hybhdbmUonof mou##, rat and human L7 mRNA Figure 3.8 In situ hybridization of human L7 at different ages Localization of human L7 mRNA in cerebellum is visualized by in situ hybridization at different ages, from 5 day to 90 years old. Molecular layer, Purkinje cell layer and granule cell layer are indicated by M, P and G, respectively. Proximal dendrites are indicated by arrows. Scale bar is shown at 100 um. 90 Figure 3.8 in situ hytirldintlon of humm L7 mRNAat dHferenl ages 91 Figure 3.9 Alignment of human and rodent L7 proteins Human and rodent L7 proteins are aligned to compare their homology and difference. The bold type letter indicates this amino acid is different from the one in the other tow species. The N-terminals of L7 form A and form B are indicated by horizontal arrows. Two Goloco domains are indicated by two open boxes. The small boxes and the roman numbers indicate potential modification sites predicted by proteomics tools. I, N-myristoylation site is only present in human L7 protein; II, protein kinase C phosphorylation site is only present in human protein; III, casein kinase II phosphorylation site; IV, amidation site; V, cAMP and cGMP- dependent protein kinase phosphorylation site. 92 GoLoco domain GoLoco domain Mouse L7 1 MAGSPDQEGFFNLLTHVQGDRMEEQRCSLQAGPGQNPESQGGPAPEMDNLMDMLVNT3GRR 61 Rat L7 1 MAGSPDQEGFFNLLSHVQGDRMEEQRCSLQAEPGQTPESQGGPAPEMDNLMDMLANT3GRR 61 Human L7 1 MAGSPDbEGFFNLLSHVQGDRMEjGQRCSljQAGPGQfrTKpQSD|PTp|EMD|SLME»1LASTbGRR 61 1 II III III IV »Form A » Form B Mouse L7 62 MDDQRVTV^SLPGFQPIGPKDGMQKRPGTLSPQPLL TPQD PAALSF RRNS SPQPQTQAP 120 Rat L7 62 MDDQRVTV MSLPGFQPIGPKDGMQKRPGTLSPQPLL 5PQD PAALSF RRNS SPQPQTQAP 120 Human L7 62 MDDQRVTVBSLPGFQPVGflKDGAbKRAGTLSPOPLLlrPQDlpTALGFRRNSBPOPPTOAP 120 I I I I V Figure 3.9 Alignment of human and rodent L7 proteins Figure 3.10 Detection of human L7 mRNA and protein by northern blot, western blot and immunohistochemical staining respectively A. Northern blot of human L7 mRNA compared to mouse L7 mRNA. 0.5ug,1ug and 2ug of human cerebellar mRNA were loaded onto the gel as labeled. lOug of mouse cerebellar total RNA was used for the mouse northern blot. B. Wesgtem blot of human, mouse and rat L7 protein. Protein extracts from P21 and adult mouse cerebellum were used as labeled. Protein extract from adult rat cerebellum was used as shown. Protein extracts from 4 year, 28 year 58 year and 90 year old human cerebella were used as shown. Protein extract from L7 knockout mouse cerebellum was used as a negative control(K.O). For each lane 10 ug of protein was loaded. Upper panel is the L7 antibody, lower panel is the Calbindin(CaBP) antibody which is used to normalize the result. C. Double labeling of human cerebellar sections with L7 and CaBP antibodies. Cy2(red) indicates L7 and CyS(green) indicates CaBP. The yellow region on the right panel shows the colocalization of L7 and CaBP. Arrows indicate Purkinje cell axons, cell body and dendrites. 94 B human mouao mousa rat human O.Suglug 2ug Ad Ad 4yr 28yr58yr90yr K.O. «# I CaBP C.-iRP L7CaBP FIgum# 3.10 Detection of human L7 mRNA and pralain by noitham blot, wastam blot and immunoblatocbamicai staining raspactivaly L7 form A(99 aa) L7 form B(120 aa) mouse rat human mouse rat human n n(% ) n n(% ) n n(% ) n n(% ) n n(% ) n n(% ) B 4 4.0 5 5.1 2 2.0 5 4.2 6 5.0 3 2.5 G 9 9.1 8 8.1 9 9.1 12 10.0 11 9.2 12 10.0 N 5 5.1 4 4.0 1 1.0 6 5.0 5 4.2 2 1.7 P 15 15.2 15 15.2 13 13.1 16 13.3 16 13.3 14 11.7 S 7 7.1 a 8.1 11 11.1 8 6.7 10 8.3 13 10.8 T 5 5.1 5 5.1 9 9.1 6 5.0 5 4.2 9 7.5 S+T 12 12 .2 13 13.2 20 20.2 14 11.7 15 12.5 22 18.3 h%/m% : 1.66 1.56 h%/r% ! 1 .53 1.46 V. Table 3.1 Amino acids composition in rodent and human L7 proteins Chapter 4 TRANSGENIC APPROACH FOR THE STUDY OF L7 MESSENGER RNA LOCALIZATION AND TRANSLATION 4.1 L7-3UTR add-back conatruct From the results presented in chapter 3, the L7 mRNA is distributed in both the soma and dendrites(and even axons) in cerebellar Purkinje cells at early developmental stages in both human and rodent. This distribution changes with age with most L7 mRNA localized in Purkinje cell bodies in adult animals and humans. To investigate whether the localization of L7 mRNA in Purkinje cells is an active and regulated event, an in vivo transgenic approach was used. From the study of the 5'ends of different forms of L7 mRNAs, we know that the sequence difference in the first exon does not alter their distribution in Purkinje cells although the time course for each form is not the same. In addition, most of the reported elements which will affect the localization of mRNAs reside in the 3'UTR. Previously, an L7 SV40 transgenic mouse was made to test whether the L7-3'UTR is important for its localization(Feng Bian, 1998). In this transgenic mouse, the L7 3'UTR was replace with a 172 bp. fragment that includes the 3'UTR and all 3' end processing signals of the SV40-3'UTR(figure 4.1). The 97 localization of the transgene mRNA is monitored by in situ hybridization(figyre 4.2). In this transgenic mouse, the hybrid of L7-SV40 hybrid mRNA was detected mainly in Purkinje cell bodies. This result has two possible explanations. Rrst, the L7-3'UTR directs the mRNA to be localized in the dendrites. Second, the SV40-3’UTR directs the mRNA to be localized in the cell body. To further demonstrate the function of the L7-3 UTR in the localization of its mRNA in Purkinje cells, another construct was made. In this construct, the L7- 3'UTR with a mutated poly(A) addition site(u4G) was added back upstream of the SV40-3’UTR in the previous construct. In this transgenic mouse, a hybrid mRNA is made which contains the full-length L7 mRNA (with a point mutation in the polyA site) and an SV40-3 UTR at the very end(with an intact poly(A) site)(figure 4.1). The distribution of this hybrid mRNA in Purkinje cells is visualized by in situ hybridization using SV40-3 UTR probe to distinguish it from endogenous L7 mRNA(figure 4.2). The hybrid mRNA was mainly localized in the cell body with a little portion in the proximal dendrites. From the perspective of transgene mRNA localization, this transgenic animal is more like the L7-SV40 transgenic animal instead of the wild type animal. One property we noticed in the add-back transgenic animal is that the hybrid mRNA has a much higher steady state level in the Purkinje cell bodies compared to the wild type L7 mRNA. The distribution of the hybrid mRNA in Purkinje cells suggests several possibilities. First, the L7-3 UTR itself is not sufficient to restore a uniform distribution of its 98 mRNA in Purkinje cells. Second, like in the previous SV40 transgenic animal, the SV40-3’UTR may have an anchor sequence that keeps the hybrid mRNA in the cell body, and the anchor sequence may override any possible element in L7 3’UTR. It should be noted, however, that several other studies have been performed using the SV40-3UTR and no such anchoring effect has been observed. Third, a wild type L7-3 UTR which is directly followed by a polyA tail is required for the uniform distribution. 4.2 Add-back-PKC construct At the time these constructs were made, we knew little about L7 form B mRNA. In addition, the vector used for making constructs was designed as an expression vector to make an untranslatable L7 form A mRNA. In this vector, all possible ATG's in all reading frames of form A were point mutated. However, now we know that this vector could still make a translatable form 8 mRNA. Therefore, in the absence of this knowledge at that time we planned to create a tagged mRNA and add a start codon to the add-back constructs using an insertion that we had previously shown not to affect the localization of the mRNA. There are two main reasons to do this. First, for the future success of these studies we will need to introduce restricted mutations, into the 3’UTR in order to determine which sequences are important for dendritic localization of the mRNA. The effect of these mutations on localization would be difficult to determine unless a tag was inserted in the mRNA so that the mutant form could be 99 identified by in situ hybridization. Second, to add a start codon would have the added benefit of conferring ribosome binding on L7 form A mRNA. This ribosome binding ability was lacking in both constructs described above. Previously, a PKC pseudosubstrate polypeptide sequence was used to generate PKC-I transgenic animal using the same vector. In that transgenic animal, the PKC transgene mRNA was evenly distributed in the cell body and dendrites just like the wild-type L7 message. This indicates that^néither the overexpressed PKC pseudosubstrate polypeptide nor the nucleotide sequence itself affects the localization of the transgene message(De Zeeuw et al., 1998). So, the same PKC sequence was inserted into the add-back construct to make the mRNA translatable(figure 4.1). When either the PKC probe or the SV40-3 UTR probe was used, the transgene mRNA was still mainly detected in the cell body of the Purkinje cells(figure 4.2). Thus we conclude that lack of ribosome binding does not explain the failure of the L7-SV40 fusion mRNAs to be transported. Also, in this transgenic animal, the steady state level of the transgene message is much higher than the wild-type L7 message. By comparing the distribution of the transgene mRNA to the ones described above and to the original PKC construct, the most likely explanations are: first, SV40-3 UTR sequence played a key role in constraining the message in the cell body; and second, as mentioned above, the position of L7-3'UTR is critical for the dendritic translocation. More in vivo experiments are still needed to test these explanations. 100 Unfortunately the in vivo transgenic animal experiments have not yet given us a clear answer about whether the L7-3 UTR by itself or in coordination with other elements is involved in the localization of its mRNA in the dendrites in Purkinje cells. Although the in vivo approach is a valuabla one for the analysis of mRNA localization, it will take more time to complete these studies. In the meantime, due to the small overall size of the L7 mRNA and especially of its 3’UTR, we have taken a parallel biochemical approach. The identification of cytoplasmic proteins that bind to the L7-3 UTR may provide dues as to the function of this region and to the identity of proteins involved in transport and/or translational control. This will be discussed in chapter 5. 4.3 Characterization of GFP transgenic animals As mentioned above, for the in vivo experiments, we would like to have a tag to monitor the localization of the transgene mRNA and to distinguish it from the endogenous L7 mRNA when limited mutations are introduced into the construct. At the same time, if the protein synthesis of the transgene can also be monitored, we would also be able to follow the translational efficiency and the localization of the protein in living cells. In order to do this, a GFP transgenic animal was generated using the same vector such that the GFP gene is under the control of the L7 promoter(figure 4.3). The GFP gene used in this construct is an S65T EGFP version which has much higher intensity of fluorescence than the wild type GFP. In addition, the GFP sequence has been mammalianized in order to obtain 101 a higher translation efficiency in the mouse. Several GFP positive lines were obtained by PCR assay of tail DNA. In order to use the GFP as a tag to monitor both mRNA and protein, the GFP transgene has to be expressed at a reasonable level so that the mRNA and protein can be easily detected. To identify the high GFP expressers, northern blotting was performed. Because the GFP gene is under the control of the L7 promoter which will allow the transgene to be specifically expressed in Purkinje cells, cerebellar total RNA samples from different lines were used. As shown in figure 4.4 A, line 50 revealed the highest expression level of GFP mRNA, and this line was kept and used for further study. In addition, the specificity of the GFP expression in cerebellum is confirmed by in situ hybridization. As shown in figure 4.4 8, when GFP probe was used GFP mRNA can only be detected in cerebellar Purkinje cells. To confirm that the GFP protein is also property synthesized in the transgenic animal, protein extracts made from wild type cerebellum, transgenic cerebellum and transgenic midbrain were used for the western blot(Figure 4.5). As expected, a 30KD GFP band was only detected in the protein extract from the transgenic cerebellum but not in the transgenic midbrain or wild type cerebellum protein extract. When the same protein extracts were used to measure the fluorescence on a fluorometer, only protein extract made from transgenic cerebellum showed an emission peak at 517nm when 484 nm was used as the excitation wavelength(figure 4.6). This result not only showed the specific expression of GFP protein in cerebellum but also confirmed the excitation and emission properties of GFP protein as 102 described In the literature. And on vibratome sections, GFP protein has been shown to be uniformly distributed in cerebellar Purkinje cells(Zhang et al., 2001b). One goal of making GFP transgenic animals is to monitor the translation efficiency, which means we have to be able to quantify the amount of GFP protein synthesized in Purkinje cells. The level ofexpression of GFP in line 50 is likely quite high as the fluorescent emission is easily detectable in whole mount cerebellum (see Figure 4.8 below). Two different approaches have been successfully applied to examine the actual quantity of GFP protein. First, a series of diluted pure GFP protein and GFP protein extract from the transgenic cerebellum were examined by westem blotting using chemiluminescent detection. The density of the GFP bands was quantified by densitometer and the GFP concentration was calculated using a standard curve constructed from the pure GFP protein(Figure 4.7). In GFP transgenic animals, the concentration of GFP protein is 0.043ng/ug cerebellar protein extract. Second, a dilution series of pure GFP protein and protein extract from transgenic cerebellum was dotted on a membrane and the fluorescent image was captured under a fluorescence microscope. The density of each sample was measured and the GFP concentration was calculated(Figure 4.7). By this method, the calculated GFP concentration in transgenic animals is 0.03ng/ug cerebellar protein extract. This is in the range of what we obtained from westem blotting. 103 One important issue in utilizing GFP transgenic animals as a tool for further study is to ensure the animal has a wild-type phenotype. The GFP animals we obtained did not show any significant abnormal phenotype either physically or behaviorally. GFP animals have similar body weight and size as wild-type animals and their development is normal. They also did not show any phenotypic abnormality of brain and cerebellum(figure 4.8). Lastly, in the GFP transgenics the marker proteins L7 and CaBP are expressed in cerebellar Purkinje cells the same as in wild-types, and Purkinje cell body, dendrites and axon show normal morphology(figure 4.9). Altogether, GFP transgenic animals provide a powerful tool for study of mRNA localization, protein synthesis in Purkinje cells, and the morphology of the subcellular protrusions of Purkinje cells, dendrites and axons(Zhang et al., 2001b). 104 Figure 4.1 Illustration of constructs for making transgenic mice L7 gene structure is shown on the top. Open or filled boxes are exons, lines between boxes are introns. Exons are labeled either inside or under the box. L7- 3’ UTR is part of exon 4 and is indicated by a hatched box. mRNA made by wild type L7 gene or by different constructs are shown below the DMA structure. In the add-back construct and add-back-PKC construct, L7-3 UTR is indicated using the same hatched box, but it contains a point mutation within AAUAAA(U-^G). PKC pseudosubstrate mini gene sequence is inserted into the 4th exon in add- back-PKC construct. 105 genomic 3'UTR WT L7 mRNA 1A or 1B ri^ T i^ T ~ i^ M po»yA tail SV40 construct 1A or 1BI E* 2 | E*3 | Ex4F:»:^:^:^:t;’;!;!;npolyA tail g SV40 3’UTR Add-back construct 1A or 1 B l exz | E»âT~Ë»4llK'nn^!?!nnnnpolyA tail Add-back PKC construct 1A or 1BI ex 2 | exs | E% ;#K?niEE3polyA tail Figure 4.1 liiuetratlon of constructs for making transgenic mice Figure 4.2 Comparison of transgene mRNA localization in different transgenic animals The localization of the mRNA of wt L7, SV40 constmct, add-back construct and add-back-PKC in cerebellar Purkinje cells are sfiown by in situ hybridization. Molecular layer is Indicated by M, Purkinje cell layer is indicated by P and granule cell layer is indicated by G. W T L7 mRNA can be seen in both soma and dendrites; in SV40 transgenic animal, mutant mRNA is constrained to the cell body; in add-back and add-back-PKC animals, mutant mRNA is mainly localized in cell body with a minor portion detectable in proximal dendrites. In add-back and add-back-PKC animals, mutant mRNA level is much higher than wt L7 mRNA. 107 . % SV40 add-back Figure 4.2 Comparison of transgono mRNA locaNaaUon In different transgenic animais 108 Figure 4.3 Illustration of GFP construct for generating transgenic mice On the top, the structure of the vector used for making construct is shown. In exon 1A, no ATG is present; ATG’s in all frames in exons 2 to 4 are mutated. Exon 16 contains 1 ATG, which makes form B mRNA translatable. Two possible forms of mRNA generated by this construct are shown below the vector structure. Form A will only make GFP protein, form B might make a fusion protein with most of L7 protein sequence at N-terminal and a piece of nonsense amino acid sequence at C-terminal. 109 vector >jC GFP //- -// ■ lh 1A IB Ex2 Ex3 Ex4 ATG TGA T Form A GFP polyA MS ATG TGA Form 8 y GFP ÎpdyA Figure 4.3 Illustration of GFP construct for generating transgenic mice 110 Figure 4.4 GFP mRNA is specifically expressed in cerebellar Purkinje cells A. Norhtem blot showing that line 50-3 is the highest GFP expresser. B. GFP mRNA is specifically expressed in cerebellar Purkinje cells visualized by in situ hybridization. I l l 21122225 l l l l l l l l B Figure 4.4 GFP mRNA Is sptclficaily expressed In cerebellsr Purkinje cells 112 Figure 4.5 GFP protein is only expressed in cerebellum in the transgenic mice GFP protein can only be detected in the cerebellar protein extract from the transgenic anima. No GFP band is detected in wt cerebellar protein extract and in mid-brain protein extract from transgenic animal. This is a typical expression pattern of proteins driven by L7/pcp-2 promoter. 113 46KO— 30KD— 21.5KD — 14.3KD— 6.5KD— Figure 4.5 GFP protein Is only expressed in cerebellum In the transgenic mice 114 Figure 4.6 Protein extract made from GFP tranagenic cerebellum showed typical GFP protein emission property Protein extract from wt cerebellum, from transgenic cerebellum and from transgenic mid* and forebrain were used for fluorescence measurement. Buffer for making protein extract was used as a control. When 484nm is used as excitation wavelength, on protein extract from from transgenic cerebellum showed emission at 517nm, indicating the typical emission property of GFP protein. 115 2 0 - I ID EM SCO 520 500 580540 EX 484 GFP S65T cerabellum WT cerabelKim GFP S65T mid-^torabrain buffer Figure 4.6 Protein extract made from GFP transgenic cerebelium showed typical GFP protein emission property r Figure 4.7 Quantitation off GFP protein in transgenic animal A. Quantitation of GFP protein in transgenic animal using westem blot. B. Quantitation of GFP protein by measuring the fluorescence of clotted samples. Standard curve constructed using pure GFP was used to calculate the concentration of sample. 117 pure GFP protein Tg ciffoMlaroxtract (ng) (ug) 0.5 1.0 2.5 5.0 10.0 20 30 40 00 GFP B GFP 2.5ng Sng 7.5ng lOng 15ng Sample Buffer WT Tg,350ug S c r* 0.974 I(0 I 4 8 12 16 GFP[ng] Figure 4.7 QuantKalion of GFP protein In transgenic mice 118 Figure 4.8 GFP aniami showed normal brain and cerebellum phenotype and GFP Is only expressed In cerebellar Purkinje cells under L7 promoter Whole mount view of wild type (A) and GFP transgenic (B ) brain. GFP is specifically expressed in cerebellum. There is no noticeable morphology change in the GFP brain. C. Vibratome section showing the specific expression of the GFP protein in the cerebellar Purkinje cells. 119 Figura 4.8 GFP aniami ahoura nommai brain and carabaiium phanotypa and GFP i# only axpraaaad in carabaüar Purfcinja caiia undar L7 promotar Figure 4.9 Purkinje cell marker proteins are normally expressed in GFP transgenic mice and Purkinje cells showed normal phenotype Purkinje cell marker proteins L7 and CaBP are normally expressed in GFP animal. The morphology of Purkinje cell body, dendrites and axon are normal. 121 CaBP L7/CaBP Figura 4.9 Purfcinju cull m arieur proteins ara normally axpraaaad In GFP transganic mica and Purlclnja calls show normal phanotypa Chapter 5 BIOCHEMICAL ANALYSIS OF THE L7-3 UTR AND PURIFICATION OF THE BINDING PROTEINS CGI-145 AND UNR 5.1 The downstream half of the L7-3 UTR interacts with multipie cerebeiiar cytoplasmic protein extracts Some in vivo experiments regarding the function of L7-3 UTR have been discussed in chapter 4. As a complement, the in vitro biochemical analysis of L7- 3'UTR has been performed to obtain some suggestions about its possible function in vivo. Previously, the L7 3'UTR has been shown to be able to form an RNA-protein complex with cerebellar cytoplasmic protein extract by EMSA, and this complex might be involved in certain in vivo function. (Feng Bian, 1998). The mature L7 mRNA has a 3' UTR of 65 nucleotides, in which the polyadenylation signal AAUAAA is located 15 nucleotides upstream of the cleavage site(figure 5.1). To determine which sequence element is involved in the formation of the RNA-protein complex, five RNA oligos were synthesized based on the 3'UTR sequence(figure 5.1). Oligo 1 covers the middle portion of the 3'UTR with AAUAAA at its end, oligo 2 contains the first 30 nucleotides from the 5’ end of the 3'UTR, oligo 3 contains the last 29 nucleotides from the 3’ end of the 3’ UTR. 123 Oligo 4 and oligo 5 are the subregions of oligo 3, oligo 5 contains the first 15 nucleotides of oligo 3 and oligo 4 contains the last 14 nucleotide of oligo 3. These five RNA oligos were first used as cold competitors in an EMSA experiment to compete with full length 3’ UTR probe for the binding to the protein components(figure 5.2). When the full-length L7-3’UTR is used as hot probe, it can form two major shifted bands on the EMSA gel, as shown in figure 5.2 band A and B. Cold 3'UTR showed significant competition for both bands at 100 fold excess, at 500 fold excess it can compete off all the binding. The 5' end of the 3'UTR which is oligo 2 cannot compete off the binding even at 1000 fold excess, the 3' end of 3'UTR which is oligo 3 can compete«pqually well as the full length 3'UTR, at 100 fold excess it showed strong competition to the binding. Oligo 1 which contains part of the sequence of oligo 3 showed significant competition to band 8 and lower competition to band A. Oligo 4 doesn’t show any competition to either band, and oligo 5 showed weak competition to band B. When these five RNA oligos were end-labeled and used as hot probes, oligo 3 is the only one that showed similar retarded bands as 3'UTR did on the EMSA gel. When oligo 3 was used as hot probe, only cold full length 3'UTR and oligo 3 could effectively compete with it for the binding. These results suggest that the downstream half of the 3'UTR (oligo 3) is responsible for the formation of the RNA-protein complex between the 3'UTR and the protein(s), and the interaction between the RNA and protein is specific. 124 5.2 Polyadenylation signal and its surrounding saquanca ara important for the formation of RNA-protein complex In order to determine whether oligo 3 is the smallest element that is responsible for the binding, a series of mutated 3' UTR probes were generated for the binding assay(figure 5.1). The first issue we wanted to address is whether the polyadenylation signal plays a role in the binding. Two probes. ApolyAm and ApolyAd were generated by changing AAUAAA sequence only. In ApolyAm U was mutated to G and in ApolyAd the whole polyadenylation signal was deleted, both of them will dismpt the CPSF(cleavage and polyadenylation specific factors) binding. When ApolyAm was used hot probe, it caused similar retarded bands as wt 3’UTR did on the EMSA gel, but one shifted band was missing when ApolyAm was used, and it also strongly competed with wt 3’UTR for the binding(figure 5.3). When the AAUAAA was completely deleted, ApolyAd could still cause mobility shift but the binding of the protein to this probe was severely reduced, and the ApolyAd probe did not compete strongly to the wt 3’UTR(figure 5.4). Because mutation or deletion of AAUAAA could effectively block the binding of the 160KD subunit CPSF to the RNA, but both probes could still form an RNA-protein complex, this suggests that most of the shifted bands revealed on the EMSA gel are not a complex between CPSF and 3’UTR. On the other hand, the dramatically decreased binding to ApolyAd suggests that either the AAUAAA hexamer or the spacing it provides are important for the binding. The second issue we considered is the surrounding sequence of AAUAAA. Five more 125 mutated probes M through AE were generated, all of them had their AAUAAA hexamer deleted in addition to the mutation or deletion of its flanking sequence(figure 5.1). AA showed weak binding ability on the EMSA gel and it can hardly compete with wt 3’UTR. AB had an aberrant binding due to unknown reasons, it poorly competed with wt 3’UTR. AC and AD had no binding ability and couldn’t compete with wt 3’UTR(figure 5.5). All these probes have deletions of AAUAAA flanking sequence. AE which had mutated AAAA to GAGA upstream of AAUAAA and mutated UUGG to AGGU downstream of AAUAAA showed no binding ability and poor competition with wt 3’UTR(figure 5.6). These results indicated that sequence AAAAGAAUAAAAGAGUUGG is important for the binding because any change of this sequence would result in losing binding ability. To confirm this, another three mutated 3’UTR probes AF, AI and AJ were made, in these three probes, the AAUAAA hexamer was kept but its surrounding sequence was changed(figure 5.1 ). All of these three probes lost most of their binding ability(figure 5.7A) and could poorly compete with wt 3’UTR(figure 5.78). This further suggests that AAUAAA surrounding sequence itself could dramatically affect the binding ability even though the wt hexamer sequence is present. When RNA homopolymer oligos were used as cold competitors, polyG did not compete with wt 3’UTR at all, polyG showed weak competition. polyA competed off a major band and polyU competed of most of the bands. Ifs not surprising that polyA could compete with wt 3’UTR because the short sequence identified has a high A%. PolyU could potentially anneal to the high A region and 126 block the binding of the proteins to the RNA and hence showed strong competition on EMSA gel(figure 5.8). This confirms that A bases in the polyA signal and in its surrounding sequence could be critical for the formation of the RNA-protein complex. As stated later in this chapter and chapter 6, one of the purified proteins, UNR, has a preferred A-rich binding site. 5.3 Several proteins are Involved in the RNA-protein complex To investigate an RNA-protein complex, on one hand we need to know the cis- element that is involved in the binding, on the other hand ifs important to gain the knowledge of the protein components that are interacting with the cis-element. In order to find out some of the information of the proteins involved in this complex, first we did a UV cross-linking experiment. In this experiment, two major bands were shown to be UV cross-linked to the probe, one of them was around 95KD and the other one was around 50KD. To examine the specificity of the result, competition experiment was performed using cold wt 3’UTR and RNA oligos. As shown in figure 5.9, wt 3’UTR and oligo 3 competed well with both bands, oligo 1 which contains the 5’end of oligo 3 competed with the 50KD band, oligo 2 and oligo 4 didn't compete with either band, oligo 5 which is the 5’ end of oligo 3 competed well with the 50KD band. This result clearly showed that these two bands are specific to the 3’UTR and oligo 3, and it further confirmed that oligo 3 is responsible for the binding as shown in EMSA competition experiment. To further confirm that these two proteins were labeled in the UV cross-linking 127 ■p ’ s -I - A: experiment, the RNA probe and protein extract mixture was UV irradiated and then loaded onto an EMSA gel, retarded bands were excised and the protein was eluted from the gel slice. Eluted proteins were then resolved on a SDS-PAGE as shown in figure 5.10. Two major complexes A and B were excised from the EMSA gel. Complex A showed 3 bands on SDS-PAGE, one is -95KD and the other is -50KD, these two bands have been shown to be specific to oligo 3, the third one which is ~70KD was not always seen in UV cross-linking experiment. Complex B mainly showed one band on SDS-PAGE, it is -50K D which runs at the same position as the 50KD protein from complex A. These confirmed that these two proteins are involved in the RNA-protein complex. 5.4 Partial purification of protein components interacting with oligo 3 As a first step to purify the proteins interacting with oligo 3, pilot experiments were done with mouse cerebellar cytoplasmic protein extracts. A general property of RNA binding protein is that it tends to be charged at its surface and binds to charged column media. So the first column we used for the purification was the weak anion exchanger, DEAE. Cerebellar cytoplasmic extract from 140 wild type mouse cerebella was loaded onto a DEAE column. The DEAE column fractions were then subjected to both EMSA and UV cross-linking assays to determin the binding activity(figure 5.11). Both the 95KD and 50KD proteins bind to the DEAE column, and the peak activity was eluted at a salt concentration of 150mM to 210mM KCI. Due to the higher salt concentration in the fractions, on 128 SDS-PAGE these proteins tend to run slower and showed a higher molecular weight compared to input sample. Figure 5.12 showed the protein profile of DEAE fractions, the peak activity is very close to the protein peak. A common matrix used for purifying nucleic acid binding proteins is heparin sepharose. Heparin is a highly sulphated glycosaminoglycan with the ability to bind to DMA and RNA binding proteins by mimicking the polyanionic structure of nucleic acids. The activity peak from the DEAE column was loaded onto a heparin column. The binding activity of each fraction was then determined by both EMSA and UV cross-linking(figure 5.13). Both the 95KD and 50KD proteins bound to the heparin column, the peak activity was eluted at a salt concentration of 450mM to 530mM KCI for both, and the 50KD protein comes out of the column at a little bit higher salt. Figure 5.14 shows the protein profile of fractions from the heparin column .Tthe peak activity is very close to the protein peak. For an intermediate step of protein purification, a hydrophobic interaction column is often used. This matrix has either an aromatic group (like phenyl-) or long hydrocarbon chains (like octyl-). When a hydrophobic environment is provided, proteins loaded onto this column can interact with the matrix according to their hydrophobicity. Activity peak fractions from the heparin column that mainly contained the 95KD protein were loaded onto a phenylsepharose column. Eluted fractions were analyzed by EMSA(figure 5.15). The binding activity peak was 129 eluted at an (NH 4)2S0 4 concentration of 120mM to OmM. At this stage, from silver staining of the fractions, a band around 95KD can only be seen in the activity peak fractions(figure 5.16). When we got to this step, the protein in the peak fractions of the HIC column was only detectable by silver staining. The MALDI-MS sequencing method that is available to us requires more substantial amounts of protein, and minimally requires that the protein can be detected by coomassie staining, this forced us to look for some other tissue sample that is sufficient to carry on more purification steps. As a close relative to the mouse, rat cerebellar cytoplasmic protein extract was shown to form a similar RNA-protein complex by EMSA and similar SOKD and 95KD protein bands were detected by UV cross-linking. So rat cerebellar cytoplasmic extract was used for the further purification. 5.5 Purification of the SOKD and 95KD L7-3’UTR-binding proteins from rat cerebellar protein extract As for the pilot experiment, rat crude protein extract was first loaded onto a DEAE column. The binding activity of each fraction was determined by EMSA and UV cross-linking(figure 5.17). The peak activities for 95KD protein are fractions 31 -33, and for the 50KD protein they are 32-34. Both were eluted at ISOmM to 200mM KCI. The activity peak is close to the protein peak as in the mouse DEAE fractions(figure 5.18). The activity peak which contains both the 130 95KD and 50KD proteins was then loaded onto a heparin column, and the binding activity of each fraction was analyzed by EMSA(figure 5.19). By UV cross-linking, both the SOKD and 95KD proteins were detected together in the same fractions. They were eluted at a KCI concentration of 450mM to 53CmM. The activity peak is still close to the protein peak as shown in figure 5.20. Then the activity fractions were pooled together and loaded onto a phenyl-sepharose column. As before, the binding activity of each fraction was analyzed by EMSA and UV cross-linking(figure 5.21). The binding activity peak of the 95KD protein was eluted at 120mM ammonium sulfate. The binding activity peak of the 50KD protein was close to the 95KD peak but it was separated. The binding activity peak was also separated from the protein peak(figure 5.22). The activity peak of the 95KD fractions were then loaded onto a high resolution mono Q column and the fractions were analyzed by EMSA(figure 5.23). The peak activity was eluted at 360mM to 460mM KCI. When oligo 3 probe was used for UV cross-linking of the activity peak fraction in the absence of RNAse, a band at -105KD was detected. After substraction of the mass of oligo3 which is -10KD, the expected size of the protein should be -95KD. When full length 3’UTR probe was used in the presence of RNAse, a band at -95KD was detected, this size should include the protein and the protected RNA(figure 5.23C). When the fractions with peak activity were examined by silver-staining on SDS-PAGE, a strong band at about 90KD was detected. So the 90KD band was excised and submitted to the Yale Cancer Center Mass Spectrometry Resource for.^^ALDIdMS assay. 131 The fractions with peak binding activity of the 50KD protein were pooled and loaded onto an SP column. The binding activity of each fraction was analyzed by UV cross-linking(figure 5.24). The 50KD protein binds tightly to the SP column and was eluted at a salt concentration of 740mM to 900mM KCI. The binding activity was highly enriched in one fraction. Then the peak fraction was loaded onto a mono Q HR column and fractions were analyzed by UV cross- linking(figure 5.25). Peak of the activity was eluted at a KCI concentration of 550mM to 600mM. Oligo 3 was then used for UV cross-linking analysis of the peak fractions in the absence of RNAse. A band at -58KD was detected. When corrected for the size of the bound oligoS, the molecular weight of the protein would be 48KD. When the full-length 3’UTR probe was used in the absence of RNAse, a band at -78KD was detected. When corrected for the size of 3’UTR probe, which is -20KD, the expected size of the protein should be 48KD. This is in agreement with the measurement obtained using oligo3 probe. When the full- length 3’UTR was used for UV cross-linking in the presence of RNAse, a band at -50KD was detected. This size should include the protein and the protected portion of RNA(figure 5.25B). In fact, a band of -48KD was observed on the silver-stained SDS-PAGE gels of the active fractions (figure 5.26). The 48KD band was excised and submitted for MALDI-MS assay. 132 5.6 The SOKD protein is the rodent homologue of human CGI-145 homologue and the 95KD protein is rat UNR The 50KD protein has been Identified by MALOI-MS and confirmed by nanospray MS/MS to be homologous to human CGI-145(accession number XM_0l2290)(figure 5.27 and figure 5.28), which is identified by in silicon analysis(Lai et al., 2000). When a TBIast search was performed using the human CGI-145 protein sequence, two pieces of truncated mouse cDNA sequences were pulled out from the database(accession number AK008946 and AK005810). By using these cDNA sequences, a 500KB mouse genomic sequence was found. Within this genomic DNA sequence, the protein-coding exons spanning -20Kb of the CGI-145 gene were identified. Several DNA primers were synthesized based on these sequences. Then cONA fragments were amplified by RT-PCR from both mouse and rat using these primers. Just one month later, a mouse cDNA sequence which is highly homologous to human CGI-145 cDNA was posted in the NCBI database(accession number BC010811). Its predicted protein sequence(accession number AAH10811) has a 95% identity to the human CGI-145 protein. This suggests that they have a highly conserved function in vivo. When the mouse CGI-145 protein sequence was subject to the CD search, no significant known domain was identified with a proper cutoff value. As of now there is no known function of CGI-145. W e have performed in situ hybridization using RT-PCR amplified fragments as probe. CGI-145 mRNA is ubiquitously expressed in embryonic day 14 mouse. However, in adult rat brain 133 the signal is detectable throughout, but is highly enriched in olfactory bulb, hippocampus and cerebellum. In the latter tissue, CGI-145 is clearly expressed in the granule cells and Purkinje cells(figure 5.29). The 95KD protein has been determined by MALDI-MS as rat UNR protein(accession number P18395)(figure 5.30). This protein has been predicted by CD search to have five CSP(cold shock protein) domains, which are RNA binding domains. It also has a 98% identity when aligned with human UNR protein. This indicates a highly conserved function of UNR between these two species. This gene has been shown to be expressed in many different tissues in rat. We have subcloned fragments of mouse UNR cDNA, and in situ hybridization showed a ubiquitous expression pattern in mouse similar to CGI-145. The UNR mRNA is enriched in the adult hippocampus and cerebellum. In cerebellum, UNR mRNA was detected in Purkinje cells and granule cells by in situ hybridization(figure 5.31). Recently, UNR protein has been shown to be involved in IRES-dependent translation regulation(Hunt et al., 1999; Mitchell et al., 2001) and in mRNA stability(Grosset et al., 2000). 134 Figure 5.1 Sequence of L7-3’UTR, RNA oligos and mutated L7-3 UTR probes On the top is the wild type L7-3 UTR sequence, polyadenyiation signal is underlined. Oligo 1 through oligoS are RNA oligos synthesized based on L7- 3'UTR sequence, polyadenyiation signal is underlined. APolyAm has a single mutation of U to G in the polyadenyiation signal, APolyAd has the deletion of polyadenyiation signal. AA through AJ have either deletions or mutations in the L7-3'UTR, deletions are represented by broken lines and mutated bases are underlined. 135 L7 3'UTR UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAACAAUAAAACACUUGGCACUAGC OLIGO 1 UGGGCCUCCCACUGGCCCCUGAAAACAAUAAA OLIGO 2 UGAGAGUUCUAGCCAUCCUGGGCCUCCCAC OLIGO 3 CUGAAAACAAUAAAACACUUGGCACUAGC OLIGO 4 CACUUGGCACUAGC OLIGO 5 CUGAAAACAAUAAAA APolyAm UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAACAAGAAAACACUUGGCACUAGC APolyAd UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAAC------CACUUGGCACUAGC AA UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAAC------CACUUGGCAC---- AB UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAAC -...... CACUUG ... Ac UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAAC------CA-...... » A d UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUG C ------CACUUGGCACUAGC AE UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGgACAC...... CACAGCUCACUAGC AF UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUG CAAUAAAACACUUGGCACUAGC AI UGAGAGUUCUAGCCAUCCUGGGCCUCCCACUGGCCCCUGAAAACAAUAAAACA...... A j ugagaguucuagccauccugggccucccacuggccccugcacacaauaaaacacagcucacuagc Figure 5.1 Sequence of L7-3 UTR, RNA oligos and mutated L7-3 UTR probes Figure 5.2 Ollgo3 can compete equally well aa full-length 3'UTR for the binding of protein components EMSA with full-legth L7 3'UTR as hot probe and cold 3’UTR or oligol through oligo 5 as cold competitors. An increasing amount of 100x, SOOx and lOOOx excess cold competitor is used as shown by triagles on to of the gel image. Free probe and shifted bands are indicated by arrows. 137 competitor - wt 3'UTR oligo 1 oiigo 2 oligo 3 oligo 4 oligo 5 A B t t free probe Figura 5.2 Oiigo 3 can compala aquaiiy twail as ffiiii-langih 3'UTR for tha binding of protain components Figure 5.3 APolyAm can form a similar RNA protein complex aa wt 3'UTR does and It can strongly compete with wt 3'UTR for the binding A. comparison of EMSA results using wt 3’UTR or APolyAm as hot probe. Arrow indicates the missing band when mutated probe is used. B. Conpetition experiment showing that cold APolyAm can be as effective as wt 3’UTR to compete with hot 3’UTR probe. An increasing amount of lOOx, SOOx and tOOOx excess cold competitor is used. 139 competitor wt 3’UTR APolyAm Figure 5.3 APolyAm can form a aimilar RNA protein complex as wt 3'UTR does and it can strongly compete with wt 3'UTR for the binding 140 Figure 5.4 APolyAd forms a signlffeantiy reduced complex and could weakly compete with wt 3'UTR for the birullng A. Comparison of EMSA results using wt 3’UTR or APolyAd as hot probe. B. EMSA showing competition results using wt 3’UTR as hot probe and wt 3'UTR or APolyAd as cold competitor. An increasing amount of lOOx, SOOx and 1000x excess cold competitors are used. 141 B % ^ competitor wt 3'UTR APolyAd » c i % ■ Figure 5.4 APolyAd forme a eIgnMIcandy reduced complex and could weakly compete wHhwfC3UTR for the binding 142 Figure 5.5 Polyadenyiation signal surrounding sequence affects the binding activity A. Comparison of EMSA results using wt 3’UTR, ApolyAd, AA, AB, AC and AD as hot probes. B. EMSA showing competition results using wt 3' UTR, AA, AB, AC and AD as cold competitors. An increasing amount of 100x, SOOx and 1000x excess cold competitors are used. Hot probe is wt 3’UTR 143 B % ^ ^ ^ competitor wt 3'UTR I ‘* * ! f t Figura 5.5 Poiyadunyfation signai surrounding ssqusncs affscts ths binding activity Figure 5.6 AE has no biniding ability and can hardly compete with wt 3'UTR A. Comparison of EMSA results using wt 3'UTR, ApolyAm, ApolyAd and AE as hot probes. B. EMSA showing competition result using wt 3’UTR and AE as cold competitors. An increasing amount of lOOx, SOOx and lOOOx excess of cold competitors are used. Wt 3’UTR is hot probe. 145 B competitor - wt 3'UTR AE Figure 5.6 AE has no biniding ability and can hardly compete with wt 3'UTR 146 Figure 5.7 Polyadenyiation signal AAUAAA surrounging sequence is important for the binding A. Comparison of EMSA results using wt 3’UTR, AF, A! and AJ as hot probes. AF and AI fail to form the major RNA-protein complex although some weak retarded bands are observed. AJ loses most of its binding ability with no sharp retarded band being detected. B. EMSA showing competition results using wt 3'UTR, AF, AI and AJ as cold competitors. An increasing amount of 100x, SOOx and 1 GOGx excess of cold competitors are used. Wt 3’UTR is the hot probe. 147 B ï AF Al AJ competitor wt 3'UTR AF Al AJ 4 H Figura 5.7 Polyadunyiation signai AAUAAA surrounging saquanca is important for the binding Figure 5.8 Poly(A) and poly(U) strongly compete with wt 3'UTR for the binding EMSA showing competition results using homopolymers poly(A), poly(G), poly(C) and poly(U) as cold competitors. An increasing amount of 100x, SOOx and lOOOx excess cold competitors are used. Wt 3’UTR is the hot probe. Poly(A) competes off one major band while poly(U) competes off most of the retarded bands. 149 competitor poiyA polyG poiyC poiyU -'-n- Figure 5.8 PolyAand polyU strongly eompsts with wt 3'UTR for the binding ISO Figure 5.9 Wild type 3' UTR and oiigo 3 strongly compete with the hot probe for the binding of two protein bands on UV cross-linking gel Wt 3'UTR and RNA oligo 1 through 5 were used to compete with hot wt 3'UTR probe in UV cross-linking experiment. SOOx excess of each cold competitors were used in the experiment. Prestained protein ladder is shown on the left of the gel image. A 90KD protein band and a SOKD protein band are shown to be competed off by wt 3'UTR and oligo 3. SOKD band can also be competed off by oligo 1 and oligo 5. 151 195KD- 126KD* 96KD- 65KD‘ 53KD- 40KD- 28KD- Figure 5.9 Wild type 3'UTR and oligo 3 strongly compete with the probe for the binding of two major protain bands on UV cross-linking gai 152 Figure 5.10 Two protein bande were Identified by UV croes-llnidng On the left panel, EMSA result showed different RNA protein complexes detected. Complexes A and B were eletroeluted. On the right panel, eluted protein components were loaded on the SDS-PAQE, straight UV cross-linking was used as control. 90KD, 70KD and 50KD protein bands were detected in complex A and 50KD protein band was detected in complex B. Arrows show the 90KD and 50KD bands. Prestained protein ladder is shown beside the SDS- PAGE. 153 complex A complex B I 65KD------53KD------ EMSA SD SfA G E Figure 5.10 Two proloin bands wars idandAad by UV cross-linking 154 Figure 5.11 Assay of binding activities of mouse DEAE fractions by UV cross-linking and EMSA A. UV cross-linking assay of DEAE fractions. Prestained protein ladder is shown beside the gel. B. EMSA result of DEAE fractions. I stands for input, F stands for flow-through. 155 FracBon» I F 8 10121314 1516 18 20 22 2426 195KD- 126KD- 90KD— 65KD— 53K D - 40K D - B Fractions I F 8 10 11 12 13 14 15 16 17 18 20 22 24 26 Figure 5.11 Assay of binding activities of mouse DEAE fractions by UV cross-llnidng and EMSA 156 Figure 5.12 Protein profile of mouse DEAE fractions A. Mouse DEAE fractions protein profile. B. Coomassie staining of mouse DEAE fractions. 157 7 6 5 0,4 ^3 2 1 0 «♦ 0 10 20 30 fractions B M I F 8 10 12 13 14 15 16 18 20 22 24 26 Figure 5.12 Protein profile of mouse DEAE fractions 158 Figure 5.13 Both 90KD and 50KD proteins bind to heparin column and are copurified A. UV cross-linking assay of mouse heparin column fractions. Prestained protein ladder is shown beside the gel. B. EMSA results of mouse heparin fractions. I stands for input, F stands for flow-through. 159 I F 33343536 37 383940 41 42 43 195KD — 126KD — 90KD — 65KD — 53KD — 40KD — 28KD — B fractions I F 33 34 35 36 37 38 39 40 41 42 43 Figure 5.13 Both 90KD and 50KD prolains bind to heparin column and are copurified 1 6 0 Figure 5.14 Protein profile of mouse Iwparin fractions A. Protein profile of mouse heparin fractions. B. Coomassie staining of mouse heparin fractions. I stands for input, F stands for flow-through. 161 0.8 I 0.6 0.4 0.2 ■0.2 tactions B fractions I F 33 34 35 36 37 38 39 40 41 42 43 m Figure 5.14 Protein profilo off mouse heparin fractions 162 Figure 5.15 EMSA of mouse HIC fractions EMSA results of mouse HIC fractions. I stands for input, F stands for flow through. A doublet can be detected in activity peak. 163 fradions F 33 34 35 36 37 38 39 40 4142 43 44 45 U Figure 5.15 EMSA off mouse HlCffrscSons 164 Figure 5.16 Protein profile of phenylsepharose column fractions A. Protein profile of mouse HIC fractions. B. Silver staining of mouse HIC fractions. I stands for input, F stands for flow-through. Protein ladder is shown beside the gel image. 165 B fractions I F 34 35 36 37 38 39 40 41 42 43 44 45 195KD 126KD Figure 5.16 Mouse protein profile of phenylsepharose column fractions 166 Figure 5.17 Binding activity assay of rat DEAE fractions by EMSA and UV cross-linking A. UV cross-linking assay of rat DEAE fractions, i stands for input. Fraction 4 is flow-through. Prestained protein ladder is shown beside the gel image. B. EMSA of rat DEAE fractions. I stands for input, fractions 2 ,4 and 6 are flow-through. 167 fractions I 4 24 26 28 29 30 31 32 33 34 36 38 40 195KD — 126KD — 95KD — 65KD — 53KD — 40KD — 28K0 — B fractions I 2 4 6 24 26 28 29 30 31 32 33 34 35 36 37 38 40 42 44 Figure 5.17 Binding activity asaay of rat DEAE fractions by EMSA and UV cross-iinking 168 Figure 5.18 Protein profile of rat DEAE column fractions A. Protein profile of rat DEAE fractions. B. Coomassie staining of rat DEAE fractions. I stand s for input, fraction 4 is flow-through. 169 r a 0 30 40 SO 60 Fficteir B fractions I 4 24 26 28 29 30 31 32 33 34 36 38 40 Figure 5.18 Protein profile of rat DEAE column fractions 170 Figure 5.19 Binding activity assay of rat heparin fractions A. UV cross-linking assay of rat heparin fractions. Prestained protein ladder is shown beside the image. B. EMSA of rat heparin fractions. I stands for input. F stands for flow-through. 171 ______fractiof» I F 5 3 S 5 56 S7 58 59 60 61 62 64 66 68 195KD- 126KD- 95KD- i 65KD- 53KD- 40KD- 28KD- B fractions I F 53 54 55 56 57 58 59 60 61 62 63 64 65 66 6768 69 70 Figure 5.19 Binding activity assay of rat heparin fractions 172 Figure 5.20 Protein profile of rat heparin fractions A. Protein profile of rat protein factions. B.^Cpomassie staining of rat heparin fractions. I stands for input, F stans for flow through 173 B fractions I F 53 55 56S758 59 60 61 62646668 Figure 5.20 Protein profile of rat heparin fractions 174 Figure 5.21 Activity assay of rat HIC fractions by EMSA and UV cross- linking A. UV cross-linking results of rat HIC column fractions. Prestained protein ladder is shown beside the gel image. B. EMSA of rat HIC column fractions. I stands for input, F stands for flow-through. 175 fracttons I F 36 40 42 44 46 48 S 0 5 2 5 4 S 8 6 2 195KD- 126KD- 95KD- 65KO- 53KD— 40KD— 28KD— B fractions I F 35 37 3941 43 4445 46 47 48 51 53 5$ $7 59 él ü Figura 5.21 AcUvNy assay o f rat HIC frid fo ra by EMSA and UV cross-linking 176 Figure 5.22 Protein profile of rat HIC column fractions A. Protein profile of rat HIC column fractions. B. Silver staining of rat HIC column fractions. I stands for input, fraction 12 is flow through. 177 I B fractions I 12 36 40 42 44 46 48 50 52 54 58 62 66 Figure 3.22 Protein profile of rat HiC column fractions 178 Figure 5.23 90KD protein was partially purified and excised for MALDI*MS A.EMSA of rat mono Q column fractions. B. Silver staining of rat mono Q fractions. I stands for input, fraction 4 is flow-througfi. Arrow indicates tfie expected protein band that was excised for MALDI-MS. C. UV cross-linking assay of fraction 23. When end-labeled oligo 3 was used as probe, RNAse treatment step was skipped, so the apparent MW should be the addition of protein and probe. When in vitro transcribed fullength 3’UTR was used, the mixture was treated with RNAse, so it showed a smaller MW. Prestained protein ladder is shown beside the gel image. D. Protein profile of the rat mono Q fractions. 179 B fractfong 4 19 21 222324 25 2627 I 4 19 20 21 22 23 24 25 26 27 ! — 4 •~ihA ..1 Vj ■-M im .m tm.m Figure 5.23 90KD protein w w peMtelly purified and excised for MALDI-MS 180 Figure 5.24 Assay of binding activity of rat SP fractions A. UV cross-linking assays of rat SP column fractions. I stands for the input, .fractions 4 and 8 are flow-through. Prestained protein ladder is shown beside the gel image. B. Protein profile of rat SP column fractions. 181 fractions I 4 8 36 37 38 39 40 41 42 43 44 45 46 195KD 126KD 95KD 65KD 53KD 40KD 28KD B Figure 5.24 Assay of binding activity of rat SP fractions 182 Figure 5.25 Binding activity assay of rat mono Q column fractions A. UV cross-linking assay of rat mono Q fractions. I stands for input, fraction 4 is flow-through. Prestained protein ladder is shown beside the gel image. 8. UV cross-linking assay of fraction 31 using either oligo 3 or full-length 3’UTR as probe. When end-labeled oligo 3 was used as probe, RNAse treatment step was skipped, so the apparent MW of the band is an addition of protein and probe. When full-length 3' UTR probe was used, the mixture was either treated with RNAse or without it. Without RNAse treatment, the apparent MW of the band should be an addition of the protein and the probe. Prestained protein ladder is shown beside the gel image. 183 ______fractions______I 4 26 27 28 2930 31 32 33 34 35 36 37 195KD— 126KD— 9 5 K D -- 65KD— 53KD—. 40KD— 28KD— B 95KD— 65KD— 53KD— 40KD— Figure 5.25 Binding activity assay of rat mono Q column fractions 184 Figure 5.26 Protein profile of rat mono Q Column fractions A. Protein profile of rat mono Q fractions. B. silver staining of the rat mono Q fractions. I stands for input, fraction 4 is flow-through. Molecular weight is indicated beside the gel image. Arrow indicates the band that was excised for MALDI-MS. 185 •111 il .J B fractions I 4 26 27 28 29 30 31 32 33 34 35 36 37 40KD Figure 5.26 Protein profile of rat mono Q Column fractions 186 ProFound - Search Result Details Détails tor rank 1 candMata In searchmmnvu-iwraieL gi|I3628010|refD(P_0I229O.2| CGI 146 pmcin [Homo np ia ul Sample 10 : UBtS, Zheng [?e»;OI Measured pepcidea : 57 Macched p«ptidt$ : 17 Min. i«qu«nc« covtrage: 30% Measured Avg/ Ccapuctd Error Residue* Kissed Mass(H) Mono Mass (ppal S t a r t to Cut Nptlde sequence 9 89.343 M 8 9 9 .4 1 4 -7 9 369 376 0 OOSVBCLR 31 2 .5 0 9 M 912.539 -32 153 161 0 AV6ZVGI£R 375.431 M 975.466 -35 94 102 0 EPGFFQSSX 3 92.429 M 992.459 -31 122 U 9 0 LSCEXIQR 1067.474 M 1067.513 -3 6 335 344 0 5S&XSEXTAK 1 149.529 M 1149.560 -2 7 U 1 129 1 RLSEEMTQR 1149.529 M 1149.560 -2 7 122 130 1 LSSENtQRR 1177.519 M 1177.551 -28 112 120 0 EHCQISm 1251.562 M 1251.599 28 173 183 0 MISCAFEOLSX 1320.732 M 1 3 :0 .7 6 6 26 82 93 0 rWHLHPAPMX 1329.679 M 1329.703 -19 35 46 0 FOACtLILSTHR 1 5 2 0 .7 '6 M 1 520 .8 0 1 -17 219 232 0 SYLLSMBIANPVTR 1 '1 2 .7 5 4 M 1 712 .7 8 2 -1 6 233 247 0 5TTC3CTQÏHHQLAK Figure 5.27 ProFound search result of CGI-145 MALOI.MS peptides 187 PmiriB & L7B4S 4(01-1565 1437J ra ■faHMdraMi ferm e QM O EQA M m fe *•!**■ a * n ■Éeoo*» 4X01-1566 12518 Yci Cd-WpoNimfemmoW N H U flO U K uBim»— ) w » m e w i a 401-1567 \50U No 401-1561 I537J No CCO-14] pooB felM B m t i i m a n w m ■MlaMfeBCai-MS 001-1569 I559J No % # f e f WWW *M a«aH ife|aatal)|*-aaaiM ■ y- - - ■ m tm * 4(01-1570 15900 Y n C d -lo S p n iü fe k M B t q p m B Q M a o K hM M apaM M HllHt(«INitclaii|» NfeNfetBMKl 401-1571 1679J Y# ■■miM uivsat m c M i a > M B 401-1573 13301 Y tt CCHWIoommWom»* i M o n u s m i 401-1572 1695J No latam ttM feifer inaiivASM Eivsat mBom,oo#|.imfe*omomm M G C » t i a > M ( 401-1574 12706 No 401-1575 1419.8 No ■taM (infefefer nrtfeoUOKQAiuat mmooo,o:fe#I.MeWfefeWoMm|fe M G C U U 2 )a M S fam ateaferiaiM inÉM 401-1576 1493.2 No n p m a q n iM A K ■0— «■«— dt«»pfe.feoig»—< f e r t p l B 401-1577 152U No On-145) IllllfeW snum uw vn mmoM,oom#IUIIfe«mfeoBfeo ■d MH m . M m I b m * 401-1571 19618 ooi»moW .m*fe»l»m"fem niOTClNiDE KTIFIED m*m««(N0femfermCIIImmmrn NnferCd-tos S l o p o N a n l B SDSPACEIk(W M 8K femm#ofeWfefe#r i M i i n M L X Figure 5.28 Summary of CGI-145 MS/MS spoctra 188 Figure 5.29 In situ hybridization of CGI-145 in aduit rat cerebellum A. Lower magnification (1x) showing that CGI-145 mRNA is detectable in granule cell layer and Purkinje cell layer. M stands for molecular layer, G stands for granule cell layer and P stands for Purkinje cell layer. B. Higher magnification (40x) showing that CGI-145 mRNA is detected in single Purkinje cell. PC stands for Purkinje cell. In both A and B, little signal is detected in molecular layer. 189 Figura 5.29 in situ hybrldiialion of C6M45 in aduit rat cerabelium 190 ProFound • Search Result Details Thn D etails for rank 1 candidate in search n im m i mns TMTTiTe gi! 137045ISPIP i 8395|IWR_RAT UNR PROTEIN gi! 112191 IplrtlSU 210 probable unr protein • n t Sii574S5|embiCAA36549.I| (X52311) unr protein (A A t-798)[Ratlusnorvegicus| Gasiple ID ; L7B95, Zhang tPassrO] Meaijred peptides : 103 Matched peptides : 22 M ;n. sequence coverage: 301 Mass iMl Kcno Mass (ppm) S t a r t To Cut Peptide sequence * 5 1 .53Z M ■351.525 3 7 1 3 7 2 1 0 AVAAPRPOR M 3 6 7 .4 7 6 21 164 171 0 EAFQFIER 9 7 6 .5 0 3 M 9 7 6 .4 8 3 21 318 3 2 6 0 EH6VXAAMR M 9 9 2 .4 7 8 10 318 3 2 6 0 EM5VIAAKR oo}.;;: M 1 3 0 9 .4 9 4 4 247 2 5 5 0 NQHOPIPGR C57.6:: M 1 0 5 7 .6 5 3 - 2 9 2 5 6 264 1 nCVDFVIPK 1 3 7 .6 1 3 M 1 1 3 7 .6 1 4 5 277 2 8 6 0 VTUECOKVR 1 4 9 .5 7 1 M 1 1 4 9 .5 7 1 0 265 274 I ElPFGOKOtK n . 5 d 5 M 1 1 7 1 .5 9 3 2 728 738 0 KITtODASAPR M 1 2 3 6 .5 1 9 12 747 758 0 GP0NSMGF6A£R 252.51: M 1 2 5 2 .5 1 4 - 1 747 758 0 6P0NSICF6AER 345.634 M 1 3 4 5 .6 3 4 4 177 186 0 EIFFKYSEFK i-' 624 M 1 3 6 5 .6 1 7 5 385 3 9 6 0 STVSFHSHSOKR 452.644 M 1 4 5 2 .6 3 6 5 65 77 0 VGOOVEFEVSSOR 46 3.73; M 1 4 9 0 .7 3 9 1 598 6 1 1 0 GEVYPFGtVGHAflK M 1 5 0 6 .6 7 3 - 1 497 5 0 9 0 OHPGFIEtAIIHOK 6 : 9 .7 3 2 M 1 6 0 8 .7 3 7 - 3 65 78 1 V600VEFEVSS0RR 6 1 " .7 2 4 M 1 6 1 7 .7 3 1 -4 744 758 1 QPR6P0NSHCFGAER M 1 6 1 7 .7 3 0 - 4 652 665 0 DQFGFXHYSVGOSK " 4 5 .7 ) 9 M 1 7 4 5 .8 2 5 -1 5 6 5 2 666 1 OQFGFtNTCVGDSXK 9 ' 5 .37; M 1 3 7 5 .9 0 9 -2 0 187 2 0 3 0 GOLEtlQPGOOVEFTIK M 2 0 1 7 .5 5 8 - 2 3 551 5 6 9 0 TKSVNGmEAOPTIYSGK 05 3.5-74 M 2 0 5 4 .0 0 6 - 1 5 300 3 1 7 0 ATHIEVISKTFQFTHEAR 130.013 M 2 1 4 0 .0 4 4 - 6 1 385 403 1 6TVSFRSR50RRFLSTVEX Figure 5.30 Profound search of rat MALOI-MS paptWaa 191 Figure 5.31 In situ hybridization of UNR in adult rat cerebellum A. Lower magnification (1x) showing that UNR mRNA is detectable in granule cell layer and Purkinje cell layer. M stands for molecular layer, G stands for granule cell layer and P stands for Purkinje cell layer. B. Higher magnification (40x) showing that CGI-145 mRNA is detected in single Purkinje cell. PC stands for Purkinje cell. In both A and B, little signal is detected in molecular layer. 192 L 3 U(71 Figura 5.31 in situ hybridlzaUon of UNR In adult rat ceraboilum 193 Chapter 6 Discussion From the previous studies, L7 gene has been shown to be specifically expressed in the cerebellar Purkinje cells. Its mRNA is distributed in both the cell bodies and dendrites and its protein is present at high level throughout the Purkinje cells. L7 protein has been shown to be a modulator of heterotrimeric G protein pathway by inhibiting GDP release from Ga subunit. Because of the distribution pattern of L7 mRNA in Purkinje cells, it is very likely that L7 protein is synthesized in both the cell bodies and dendrites. In dendrites, the synthesis of L7 protein may be locally regulated in response to external stimuli. Right now, people still do not know how the L7 protein synthesis is regulated in the dendrites, and to what extent local L7 protein synthesis would change the local L7 protein concentration in dendrites. Although local protein synthesis has been shown to play an important role for some other proteins, the physiological significance of L7 protein synthesis on dendrites remains unclear. In the previous chapters, I have provided some new evidence that the expression of L7 gene is regulated both at transcriptional level and possibly at posttranscriptionai level. In the following paragraphs, I am going 194 to discuss these results, the Implications of these results and the conclusions that can be reached. 6.1 The distribution of L7 transcripts in cerebellar Purkinje cells is developmentaily regulated We have shown here the conservation of the L7 gene between rodent and human. This conservation can be seen from the structure of the gene organization, the alternative mRNA forms, the localization of the mRNA in cerebellar Purkinje cells as well as In their dendrites, and the homology of the encoded protein. At the ONA level, both rodent and human L7 genes have four exons. Among them, the last three exons exhibit high homology(81%). Due to the alternative usage of the exon I s in both human and rodent, exon IB is consen/ed but exon 1A shares no homology. In the upstream promoter region, several regulatory cis-elements are also conserved(eg. RORa response element). These conserved elements imply similar regulatory mechanisms used by both human and rodent. As we showed in chapter 3, a choice of two alternative exonl's has been adopted by both human and mouse. In the mouse, the expression of these two forms seems to be related to the developmental stage. Previously, using a probe that cannot distinguish between the two forms of transcripts. It has been shown In mouse that at an early stage a higher fraction of transcripts is detected in 195 dendrites than in adult(Wanner et al., 2000). Here we extend this observation by showing that both of the alternative transcripts are present in both cell bodies and dendrites in mouse cerebellar Purkinje cells. Form B has a more uniform distribution in dendrites at a time point between P7 and P14 and the expression level of form B is also high during this period. FomwA seems to reach its peak level around P14. At P7 form A is mainly localized in the cell body, and by P14, form A shows a uniform distribution in dendrites. At P21, form A keeps its high mRNA level both in cell bodies and in dendrites while the level of form B mRNA in dendrites decreases. With increasing age, the global level of both form A and form B decreases. In adult animals, the level of both formA and form B in dendrites is much lower than that at early developmental stages. Interestingly, when we examined human L7 mRNA localization in cerebellar Purkinje cells, a similar pattern was observed. For these experiments, a probe was used that cannot distinguish between form A and form B. A uniform L7 mRNA distribution in both cell body and dendrites was detected at 5 days, a developmental stage which is equivalent to 1 to 2 weeks in mouse. But, in older human most of the transcripts are confined in the cell body. Due to the availability of the human tissue samples, for each age we only used the tissue from one individual. But all the tissues we used in these experiments have no abnormality of cerebellum. So we expected that the results obtained from these tissues should be able to reflect the actual situation. Especially, 5 day and 1 year old 196 tissues represent two different individuals at tfieir early developmental stage, and they all showed similar L7 mRNA distribution. Also, for all the ages we tested, the distribution of L7 mRNA showed a gradual change which is consistent with what we saw in the rodent, so it would tie reasonable and easier to explain it as a conserved feature of L7 mRNA between rodent and human. These results clearly indicate that the translocation of L7 mRNAs into dendrites is developmentaily regulated, and it is consen/ed in mouse and human. Furthermore, the time point during which more L7 mRNA is localized in dendrites is compatible with the stage of synaptogenesis. This implies that L7 protein synthesis in dendrites may be involved in synaptic plasticity. Because form B transcript is localized in dendrites at an earlier time point than form A, it indicates that form B protein is required at an earlier stage during dendritogenesis, and form A protein is required at a later stage. 6.2 Function of L7 protein The most distinctive stmctural feature of the L7 protein is that it has the GoLoco motif. Recent studies have shown that GoLoco motifs in RGB 12, RGS14 , AGS 3, Rapi GAP and LGN serve as a GDP dissociation inhibitor(GDI) (Bernard et al., 2001; Kimple et al., 2001; Natochin et al., 2001). The difference between form A and form B L7 proteins is that L7 form B has two GoLoco motife whereas L7 form A has only one complete GoLoco motif. Right now, it is not d ea r what the functional difference might be as a result of this different number of GoLoco 197 motifs. Natochin and colleagues showed that with one GoLoco motif, L7 form A inhibits the release of GDP from the Ga subunit(Natochin et al., 2001). However, in an earlier study Luo et al had argued that L7 protein promoted rather than inhibited GDP release from Go(Luo and Denker, 1999). Natochin countered this by claiming that the other group had inadvertently introduced two mutations into their L7 expression construct. The first error, they argued, was the introduction of a second GoLoco domain which we now know (but they could not have known at the time) to be a real characteristic of Form B. The other difference was a substitution of a critical amino acid (see chapter 1) (Natochin et al., 2001). In this regard, RGS12 and RGS14 contain only one copy of the GoLoco motif, whereas AGS3 and LGN contain four GoLoco motifs, but they all displayed inhibition of GDP release from Ga(Natochin et al., 2000; Natochin et al., 2001). So even though Luo’s L7 construct has two GoLoco motifs, it is still unlikely that this difference would explain the opposite effect of L7 on GDP binding in the two reports. Whatever the explanation, it seems most likely that the effect of L7 should be to inhibit GDP release from Govo. As stated by Natochin et al., L7 and other Goloco motif containing proteins can compete with Ggy subunits for the binding of GDP-bound G a subunit, and this will activate the G ^ signaling pathway in the absence of the ligand. Because the binding of L7 to G a inhibits the GDP dissociation, the activity of G a subunit is inhibited. 198 One evidence that multiple GoLoco motifs in one protein may function differentially came from a deletion analysis of AGS3(De Vries et al., 2000a). In AGS3, the fourth GoLoco motif at its C terminal seems to be a major contributor to the interaction between AGS3 and Ga. Therefore, it is possible that the two forms of L7 may have unique functions due to the difference in number of GoLoco motifs. In addition, because L7 is a relatively small protein, its sole function might be to give input into the heterotrimeric G protein pathway instead of working like RGS12 or AGS3 which can also function as scaffold proteins. But at the same time, it is important to point out that the expression level of L7 is extraordinarily high in Purkinje cells and so it could have a dominant effect in terms of the GoLoco motif function when compared to other GoLoco motif- containing proteins. In spite of this possible dominant effect in normal animals there are other GoLoco motif-containing proteins in cerebellar Purkinje cells, for example RGS14(Grafstein-Dunn et al., 2001). These might compensate for the loss of L7 protein and help explain why there is no obvious phenotype in L7 knockout animals. As stated in chapter 3, the L7 protein contains several potential modification sites. It is likely that the modification of L7 protein would affect its interactions with Go. This is even more interesting when we look at human L7 protein. Human L7 protein is highly homologous to its mouse version. The property that most distinguishes human L7 protein from that of rodents is its more potential 199 modification sites. Both human and rodent have conserved PKA/PKG phosphorylation site(RRNS) (Feramisco et al., 1980; Glass et al., 1986; Glass and Smith, 1983) and amidation site(QGRR) (Bradbury and Smyth, 1987; Kreil, 1984). In addition, human L7 protein has two N-myristoylation sites(Grand, 1989; Towler et al., 1988), one PKC phosphorylation site(Kishimoto et al., 1985; Woodgett et al., 1986) and two more casein kinase sites(Pinna, 1990). These extra modification sites give human L7 protein more ability to be regulated, and the regulation of Ga by human L7 protein could be even more finely tuned. For example, the N-myristoylation sites could potentially allow human L7 protein to be anchored at certain membrane areas to function locally. So far, it's still unclear how L7 protein is modified in vivo, and how its activity is regulated. But considering its two different forms and the variety of possible modifications, one could expect L7 protein to play an important role in regulating the G protein associated signal pathway. 6.3 Is the L7-3'UTR involved in the translocation of its message? The L7-3’UTR has been examined for its ability to translocate L7 mRNA into dendrites. As shown in chapter 4, no conclusive result has been obtained. On the one hand, when the L7-3’UTR was replaced with the SV40-3’UTR, a confined distribution pattern of L7 mRNA in the cell body was obtained . It seemed that the 3 'UTR was necessary for keeping a uniform distribution of L7 mRNA both in the cell body and in dendrites. But on the other hand, when the L7-3’UTR was added 200 back upstream of the SV40-3 UTR, it only partially recovered the translocation of L7 mRNA into dendrites. This apparent paradox could be explained by the following possible reasons. First, it is possible that the L7-3'UTR itself is not sufficient to direct the localization of L7 mRNA in dendrites. It may need extra sequence from the coding region or even from 5' UTR to work in concert to localize the mRNA in the dendrites. All of these sequences were, in fact, present in all of the L7-SV40 hybrid constructs. However, because the L7 mRNA is a short molecule, it is also possible that the overall structure of the molecule is important to work as a zipcode to localize the mRNA in dendrites(Jansen, 2001). ASH1 mRNA provides such a model for mRNA localization(Chartrand et al., 1999: Gonzalez et al., 1999). So when the SV40-3’UTR is fused with the L7 mRNA, it may change the overall structure of the molecule and hence disrupt the zipcode. Second, it is possible that the SV40-3 UTR has an anchor sequence keeping the mRNA in the cell body. While formally possible this is unlikely since the SV40-3'UTR has been used in many studies in which hybrid mRNAs carrying this sequence showed normal localization in subcellular processes(Blichenberg et al., 1999). An effective way to test this possibility is to use another 3’ UTR from a different mRNA that is known not to be translocated in dendrites. Third, the L7- 3’UTR may be required to be immediately upstream of the polyA tail in order to function as a translocation signal. To test this possibility we need to insert the SV40-3’UTR upstream of the L7-3 UTR so that the SV40-3 UTR will not interfere with the interaction between the L7-3 UTR and possible trans-acting factors. 201 Another factor that we need to consider is the possible stabilizing ability of the SV40-3’UTR. This could explain why an elevated transgene expression level was observed in the L7-3 UTR add-back animals. Other experiments that would help to understand the function of L7-3 UTR in the transport of L7 mRNA into dendrites carv be performed by using L7 knock-out animals. For example, mutations can be introduced into L7-3UTR in the transgenic construct and then put this construct into L7 knock-out animal to examine whether the transgene mRNA is transported in cerebellar Purkinje cells. The advantage of using L7 knock-out animals is that it would be easy to analyze the transgene mRNA by using L7 probe because there is no endogenous L7 mRNA. When we talk about the transport of the mRNA in dendrites, there is always a question of whether certain mRNA gets into dendrites by a passive manner. For example, is it possible for L7 mRNA to get into dendrites by simple diffusion? Or is it possible that L7 mRNA moves with the flowing of the cytoplasm and gets into the dendrites? One important reason for this concern is* that L7 mRNA is a relatively small molecule. But the answer is almost certainly negative. The study of a small RNA called SCI provided a good example to elucidate that RNA transport is an active process regardless of the size of the molecule. BC1 is a short RNA polymerase III transcript, that is only 152 nucleotides in length, one 202 third of the length of the L7 mRNA. When the full-length BC1 mRNA Is injected into the cell body of sympathetic neurons in culture, it is specifically delivered to dendrites. When the BC1 RNA is divided into a 5' half and a 3’ half and then injected into sympathetic neurons, the 5’half of 62 nucleotides is translocated into the dendrites to a similar extent as the BC1 full-length RNA. In contrast, the 3’ half of 60 nucleotides remains in the cell body after injection without any significant diffusion. Now we know that a dendritic targeting cis-element is localized within the 5 end of the RNA(Muslimov et al., 1997). Furthermore, from the dynamic point of view, the much slower diffusion rate would be an insurmountable barrier for the mRNA to get into the highly branched dendrites like the one Purkinje cells have. 6.4 The L7-3’UTR contains a cis-alement that is conserved between species By in vitro biochemical assay, we determined a 17 nucleotide element in the L7- 3’UTR, which is responsible for the formation of an RNA protein complex. This sequence has been shown to be consen/ed between rodent and human. The 3’ UTR is an important location for many mRNA processing events. So far the 3'UTR in other sytems has been shown to be involved in polyadenylation, RNA stability, subcellular localization, transcription and translational regulation. The L7 mRNA has a very short 3'UTR which contains a typical polyadenylation signal AAUAAA. No other cis-element was identified by sequence analysis. When cerebellar cytoplasmic protein extract was used, several RNA-protein complexes 203 were detected using the 3’ UTR as a probe by EMSA. Our original goal was to identify the major complexes to determine what the necessary cis-elements are, and what protein components are involved. We expected to obtain some Information about whether the 3' UTR and its interacting factors are involved in RNA translocation, or possibly to define other important functions of the L7- 3'UTR by identifying the proteins that interact with it. By deletion and mutation assays, we showed that the polyadenylation signal is required for the formation of the major complex and its absence showed a detrimental effect on the RNA-protein complex formation. The element defined here is a purine-rich sequence and it flanks the polyadenylation signal. It is reported that cytoplasmic CPSF, which is homologous to nuclear CPSF, binds to AAUAAA in the cytoplasm and works together with CPEB(cytoplasmic polyadenylation element binding protein) to regulate the length of the polyA tail (Dickson et al., 1999). It would be interesting to test whether the cytoplasmic CPSF is one of the components in the RNA-protein complex detected by EMSA if the cytoplasmic CPSF is present in cerebellar Purkinje cells. And when we look at the EMSA result using polyAm as a hot probe in which the key residue of the polyA signal has been mutated, the major complex is resenred but one band is lost. From previous report, we know that the mutation of U->G in polyA signal would block the specific binding of CPSF to the polyA signal, so the missing band in the EMSA experiment using polyAm as hot probe may be due to the inability of 204 the CPSF to bind to this probe. Even if there is no cytoplasmic CPSF present in the cerebellar Purkinje cells, there is still a chance for the nuclear CPSF to be present in the protein extract. Because of the consen/ation of the defined element between rodent and human, and because rat cerebellar cytoplasmic extracts can also form the same RNA-protein complex with the mouse 3’ UTR, one would expect a conserved function of this complex. 6.5 L7-3’UTR-interacting proteins Here we identified two proteins that interact with the L7-3’UTR, one is CGI-145, and the other is UNR protein. Each of these two proteins has.the capability to be UV cross-linked to the L7-3 UTR under our experimental conditions. Competition experiments have indicated that the binding of these two proteins to the L7- 3'UTR is specific. Also these two proteins could be only part of the protein portion in the RNA-protein complex as shown by both UV cross-linking and EMSA assays. 6.5.1 CGI-145 is ubiquitous and highly conserved Mammalian CGI-145 was first identified by comparative proteomics analysis(Lai et al., 2000). We have examined the expression pattem of CGI-145 in various mouse tissues. The results suggest that the expression of CGI-145 is ubiquitous. In cerebellum, CGI-145 has been found to be expressed in both granule cells and Purkinje cells by in situ hybridization. The identity between human CGI-145 205 protein and its mouse homologue is 92%. This high homology implies that CGI- 145 has a conserved function at least in human and rnouse. Protein sequence analysis revealed no significant homology to any known domains or motifs. So far there is no reported function with respect to this protein. Considering the ubiquitous expression pattem and the highly conserved protein sequence as well as the ability to interact with mRNA, CGI-145 may be involved in some fundamental events in vivo. 6.5.2 UNR, an RNA binding protein, binds specifically to RNA with high affinity The unr(upstream of N-ras) gene was first isolated during the study of N-ras, because it is closely linked to the N-ras gene with an intergenic sequence of only 150bp(Boussadia et al., 1993; Jacquemin-Sablon and Oautry, 1992; Jeffers et al., 1990; Nicolaiew et al., 1991). This genetic organization has been noticed in mouse, rat, guinea pig and human. The unr gene has been shown to be ubiquitously expressed in various tissues in mouse and human, and high level unr transcripts were detected in brain by northem blot(Jeffers et al., 1990; Nicolaiew et al., 1991). It has been shown that the unr gene has three different forms of transcripts due to differential usage of the 3’UTR and most of them are present in many tissues with different abundance. During our purification of the UNR protein, a doublet was obsenred by silver staining and by UV cross-linking. This was also observed by Hunt et al during their purification. The difference of 2 0 6 the two bands is about 3KD(Hunt et al., 1999). By sequence comparison, UNR protein has been found to contain five CSD(cold-shock domain) repeats wfiich are nucleic acid binding domains(Doniger et_al.^M992; Jacquemin-Sablon at al., 1994). It has been shown that UNR protein has" very high affinity to single stranded DNA and RNA, but low affinity to double-stranded DNA. Recently, by using the SELEX method, the UNR protein preferred binding sequence was identified(Tiiqueneaux et al., 1999). This conserved binding sequence was divided into two groups. The GU group has a core sequence of AAGUA/G and the AC group has a core sequence of AACG/A. The flanking sequence of both the core sequences shares one common feature which is that they are purine- rich, especially A rich. Mutation of the core sequence decreases the binding activity, whereas introduction of pyrimidine into the flanking sequence has variable effects on the binding, either decreasing it or causing no change. In our identified L7 element, an AC core sequence Is present and -70% of the bases are As. This fits well with the favorable binding sequence of UNR. The defined cis-element by our biochemical analysis contains UNR optimal binding sequences as shown below. MOUSE AAAACAAUAAAACACUUGG RAT AAAGUAAUAAAAUACUUGG HUMAN AAAAGAAUAAAACACUUGG For both the G U or AC core sequences, UNR has a similar Kd value which is about 10 to 20 nM. The sequence requirement and Kd constant suggest the 207 binding of UNR to RNA is specific and strong. This is compatible with the competition experiment using L7 3’UTR as probe. 6.5.3 The UNR protein is involved in translation and mRNA decay, and is essential for survival By protein sequence analysis, the human UNR protein showed a 98% identity to its mouse homologue. This strongly indicates a highly conserved function of this protein. A UNR knockout mouse has a lethal phenotype suggesting that the unr gene is essential(Boussadia et al., 1997). The cold-shock protein domains which are present in the UNR protein have been found in many other proteins, including cold-shock proteins in bacteria and some eukaryotic proteins. The cold-shock domain containing proteins have been thought to function as a regulator of translation in eukaryotes and function as an RNA-chaperone in bacteria. In Xenopus, one CSD containing protein FRGY2 has been shown to specifically bind to certain mRNAs and repress translation in developing oocytes. But during oogenesis, the mRNAs are released from the RNA-protein complex and translated. The function of CSD in FRQY2 has been shown to provide the specific recognition between mRNA and the FRGY2(Bouvet et al., 1995; Bouvet and Wolffe, 1994). In C.elegans, Lin-28 exon2 encodes a region which is homologous to CSD. Lin-28 protein has been shown to regulate its downstream gene expression posttranscriptionally(Moss et al., 1997). Other 208 CSD-containing proteins in eukaryotes include MSY2, DjY1, p50. MSY2 is a mammalian homologue of Xenopus FRGY2, it is coincident with the stored mRNA at one cell stage embryo and it is not detected at later two cell stage when the bulk maternal mRNA is degraded(Gu et al., 1998). DjYI is a protein expressed in Dugesia japonica in regenerating tissues. DjYI has been thought to be involved in the posttranscriptional regulation of regeneration-related genes(Salvetti et al., 1998). All these proteins have been suggested to be involved in the regulation of translation of certain mRNAs. Recently UNR protein has been shown to be involved in the stimulation of 1RES- dependent translation(or cap-independent translation). IRES(intemal ribosome entry site)-dependent translation has been found both in viral gene translation and in cellular gene translation(Martinez-Salas et al., 2001). IRES-dependent cellular gene translation has been demonstrated to function in apoptosis and in stress. Hunt et al showed that UNR protein together with PTB(polypyrimidine tract-binding protein) stimulate IRES-dependent translation of human rhinovirus(Hunt et al., 1999). Another more interesting evidence came from the study of the cellular Apaf-1(apoptotic protease-activating factor 1) IRES- dependent translation. Mitchell et al showed that UNR and PTB together stimulate the translation of Apaf-1 via 1RES both in vitro and in cell lines. Furthermore, they showed that UNR but not PTB directly binds to the 1RES, but in the presence of UNR, PTB binds to the IRES(Mitchell et al., 2001). This result 209 is consistent with the finding that UNR protein is associated with ER in the cytoplasm(Ferrer et al., 1999; Jacquemin-Sablon etal., 1994). By monitoring the protein synthesis, one interesting phenomenon has been observed in some dendritic localized mRNAs. Pinkstaff and colleagues showed that IRESes were present in five dendritically localized mRNAs, ARC, CaMKIIa, dendrin, MAP2 and neurogranin (RC3) (Pinkstaff et*-al., 2001). For these five mRNAs, both cap-dependent and IRES-dependent (cap-independent) translations are used in neurons with a greater contribution of IRES-dependent translation in dendrites. So it would be very instructive to know whether IRES- dependent translation in dendrites is activated upon external stimuli to complement the insufficiency of the cap-dependent translation. This would also be consistent with the observation of some upregulated protein synthesis at synapses when stimuli are received(Scheetz et al., 2000). 1RES element was originally discovered in picomavirus(Jang et al., 1988), later on It was also identified in cellular genes(Martinez-Salas et al., 2001). In virus, 1RES has been shown to have conserved secondary structures. Few cellular IRESes have conserved secondary structure(Le and Maizel, Jr., 1997), and most of them share poor homology of the primary sequence or the secondary structure. Recently, short cis-elements with 1RES activity in cellular mRNA have been identified(Owens et al., 2001). The 5’ leader sequence of the five dendritic 210 mRNAs mentioned above have shown 1RES activities, but no specific 1RES sequence has been identified yet. It would be interesting to test whether L7 mRNA contains such an 1RES active element and whether UNR protein plays a role in it. But, because UNR protein binds to the 3’UTR of L7 mRNA while 1RES should be upstream of the translation start codon, it is still difficult to predict whether UNR protein would facilitate an IRES-dependent translation of L7 mRNA in vivo. Apart from the stimulatory effect of the UNR on the 1RES dependent translation, it has been shown that UNR is also an important player in stabilizing mRNA by blocking deadenylation. mCRD(major protein-coding region determinant) has been identified as a destabilizing element of mRNA in the coding region of the c- fos gene. mRNAs containing mCRD would be rapidly degraded. A protein complex has been shown to interact with the mCRD to stabilize the mRNA. Proteins in this complex have been identified as PABP(poly(A) binding protein), PAIP-1 (PABP-interacting protein 1), UNR, NSAP1 and hnRNP D. Overexpression of UNR, PAIP-1 and NSAP-1 blocked the deadenylation of the mRNA. The proposed working model for this complex is that this complex stabilizes the mRNA before translation. During translation, ribosome transit would disrupt this complex and cause mRNA rapid degradation after translation(Grosset et al., 2000). This result suggests UNR is involved in the coupled translation and mRNA turnover event. 211 Considering these facts of the UNR protein and the interaction twtween L7 mRNA and UNR protein, it would be interesting to know whether the binding of UNR protein to L7 mRNA affects its translation or stability or both. As a matter of fact, both L7 mRNA and protein have a high steady state level in Purkinje cells. Does the binding of a UNR-containing complex to L7 mRNA play a role to stimulate the translation or to stabilize the mRNA? It also would be very interesting to know whether L7 mRNA can be translated in a cap-independent manner in dendrites. Right now, we still do not know whether UNR protein is present in the dendrites in Purkinje cells. It will also be interesting to find out whether this RNA-protein complex contributes to L7 protein synthesis and mRNA stability in dendrites in response to external stimuli. And there might be even more possibilities when CGI-145 is considered. Several experiments can be designed to test these possibilities. First, to confirm the specific interaction between the L7 mRNA and UNR protein, we can overexpress the UNR protein in bacteria and use it to do EMSA and UV cross- linking with L7-3 UTR probe. Second, we can use bacteria expressed UNR protein to make antibody. UNR antibody will allow us to perform the supershift experiment on the EMSA gel to further confirm the interaction between the L7 mRNA and the UNR protein. We can also use UNR antibody to determine the distribution of UNR protein in cerebellar Purkinje cells. By using UNR antibody, we can perform immunoprécipitation experiment so that we examine what other 212 protein components are present In this RNA-protein complex formed between L7 mRNA and UNR protein. Third, to test whether the UNR protein is involved in the regulation of translation, we can use in vitro translation kit to translate L7 mRNA in the presence or absence of UNR protein and compare their translation efficiency. In this experiment, an internal controfj's needed to nonnalize the results between the UNR+ and UNR- group. Fourth, to test the possibility that L7 mRNA contains an 1RES lelment, L7 cONA can be fused to a reporter gene and cotransfected with 4E-BP1 (Pinkstaff et al., 2001), which can block the cap- dependent translation, into cultured cells. If the reporter gene is expressed, then we know that cap-independent tranlstion is involved. An internal control is also required for this experiment to normalize the reduction of translation caused by the expression of 4E-BP1. 6.6 Conclusions Base on the experiments presented in chapter 3 , 1 conclude: 1) L7/pcp-2 mRNA has two different forms in rodent and human due to the differential usage of the first exons; 2) the expression and distribution of L7 mRNA are developmentaily regulated in cerebellar Purkinje cells; 3)Two forms of L7 proteins can be predicted based on the mRNA sequences, which contain the functional GoLoco motif(s) in mediating the interaction between the L7 protein and Ga subunit. 213 Based on the experiments presented in chapter 4 ,1 conclude; 1)L7-3’UTR is involved in the localization of L7 mRNA in cerebellar Purkinje cells; 2)The utilization of GFP reporter gene in the transgenic constructs can facilitate the study of mRNA transport and the study of translational efficiency of the transgene. 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