Regulation of Nuclear Transport and Mitosis by Ran GTPase Ting Chen Changsha, Hunan, P., Xiamen University, 2001 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Microbiology University of Virginia January, 2007 linn fee p~ maA UMI Number: 3238148 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.
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Box 1346 Ann Arbor, MI 48106-1346 Abstract One of the hallmarks of the eukaryotic cell is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments is mainly regulated by the small GTPase, Ran. The nucleotide exchange factor RCC1 catalyzes formation of RanGTP, whereas the hydrolysis of RanGTP is stimulated by RanGAP1 and RanBPI. Because RCC1 is chromatin-bound throughout the cell cycle while RanGAP1 and RanBPI are cytoplasmic, the concentration of RanGTP is high near the chromatin.
This RanGTP gradient is essential for many cellular processes, including nuclear transport during interphase, mitotic spindle formation, and nuclear envelope assembly during mitosis. During the initial phase of my project I examined the nucleocytoplasmic shuttling of an unusual protein called JAZ, which does not contain classical dsRNA binding domains but instead binds RNA with high affinity through C2H2 zinc fingers. I showed that JAZ is a nuclear protein at steady state but is highly dynamic within the nucleus and undergoes nucleocytoplasmic shuttling. JAZ associates with Exp-5 in the presence of RanGTP and a hairpin RNA, and nuclear export of JAZ requires Exp-5.
However, JAZ also binds to ILF3, in an RNA-independent manner, and JAZ and ILF3 can form a heteromeric complex with Exp-5 and RanGTP. Unlike ILF3, JAZ does not contain a classical NLS. In principle it could diffuse passively through the nuclear pores, and | showed that import is indeed independent of soluble transport factors. However, import ii is inhibited by wheat germ agglutinin and by low temperatures, which do not inhibit passive diffusion.
In the second phase of my project, I discovered a unique modification on RCC1, in which the N-terminal Ser/Pro residue of mammalian RCC1 is methylated on its œ- amino group. A methyltransferase activity for N-terminal methylation of RCC1 is present in soluble nuclear extracts from HeLa cells. Methylation-defective mutants of RCC1 are unable to bind as effectively as wild type protein to chromatin during mitosis, — which results in supernumerary centrosomes and spindle formation defects. These defects are additive to those caused by a mutation that disrupts exchange activity, and may result from decreased binding to DNA.
Coupling RCC1 to histone H2A, to force chromatin attachment, reverses the mitotic defects. ili Dedication This dissertation is dedicated to my mom Guihua Li and my husband Mingda Hang. Without their constant love and support, this dissertation would never have been possible. iv Acknowledgements First, I owe my deepest thanks to my dissertation mentor, lan Macara.
lan's enthusiasm for science, breadth of knowledge, willingness to share his time, and high stands of scientific merit made great impact upon my graduate study. This work would not have been possible without his guidance and support. I also thank all members of my dissertation committee— Ann Beyer, Mitch Smith, Lucy Pemberton, Marty Mayo, and Lou Hammarskjold. Their advice was always helpful, and they were extremely supportive throughout my graduate studies.
I’d like to thank all members of the Macara laboratory, past and present, for their support to my graduate study. In particular, I thank: Amy Brownawell, Kendra Plafker, Scott Plafker, Quansheng Du, Lin Gao, Greg Riddick, Christine Schaner-Tooley, Yi Qin, Julia Dorfman, Claudia Low, Brandon Kremer, Chris Capaldo, Huaye Zhang, Xinyu Chen, and Jim Crawford. I hope that they enjoyed our time together as much as I did. I am grateful to many individuals within the Center for.Cell Signaling for their help: Dr.
Pemberton, and Dr. I also thank Mary Beth, Patricia Arkhurst, Drew Thomas, Flora Terry, and Karen Neale for their daily assistance in the Center for Cell Signaling. I would like to thank Martha Campbell and Sandy Weirich for their kindness and help throughout my study within the Microbiology Department. Finally, I would like to thank my family for all their love and support.
Without them this would not be worthwhile. Table of Contents ABSTRACT | I DEDICATION m ACKNOWLEDGEMENTS IV TABLE OF CONTENTS M LIST OE FIGURES VII LIST OF ABBREVIATIONS : xX CHAPTER I. GENERAL INTRODUCTION 1 RANGTP AND NUCLEAR TRANSPORT. 0 HH TH nh ng TH ng Tu HT TT TH ng g0 2 The Nuclear Localization SiBTIQÌ.
«cty TH HH TH TT 1T 016 KT 4 The Nuclear Pore COTHDÏ6X. Ăn HH HH HH Hà. 8 The Nuclear Transport ReC€jÍOFS. LH HH HH HH HH HH 01H HH 13 ý, 8.
25 Detection of RanGTP Gradients on Mitotic Chromosomes in vitro and in VÌVO.c 26 Function Of Ran in Spindle FOTiidfiOH. ch HH ng HH nh 28 Function of Ran in Centrosome FOTI[ÍO. cà ch HH HH HT HH HT Là HH. THE NUCLEOCYTOPLASMIC SHUTTLING OF JAZ 36.
(5G <4 H91 T9 10T Ầ TT HT TT T9 040041.00 4106 41 JAZ is a nucleocytoplasmic shuttling DFOÍGH. ch HH HH ng HH r 41 Nuclear export of JAZ is dependent on Exp-5 in both digitonin-permeabilized cells and intact cells. 44 JAZ binds ILF3 independently of dsRÌA. HH HH HH TH He Ho TH n0 000k.
53 JAZ, ILF3 and Exp-5 can form heterotrimeric COTIDÍGX. Ăn HH HH ghg 62 JAZ is imported into the nucleus by a nonclassical meCh@HÌSTH. cành re, 65 DISCUSSION 00105777. 74 MATERIALS AND METHODS.
HH HT Họ. ch TL HT 0 016 2H 0.0301 11m 76 Cloning, antibody and recombinant protein €XDT€SSỈOH,.à s cnhnHnnHnH Hà H Hg 76 Yeast dihybrid and conjugation (5S/3. Gà LH nu HH TH TH g2. 77 Cell culture, transfection, and pFOCGSSÍH.
ch HH1 2 1 HH HH rệt 77 RNA binding assays .2 200 nẼnBẼn0Ẽn0nẺ000 0n. 79 MICrOINJCCEION EEEEEEEETE Sổ 0n CỐ ca na cố cố nan. 80 FateroKar yon fusiOn ASSAYS 0000000808080 060 nh. 81 Permeabilized cell transport GSSÿ.
ST HH HH HH HH TH Tà HH Hi TH 001011. N-TERMINAL œ-AMINO METHYLATION OF RCCI 83 9): am na. (HH TH TH TH TH TT HT TT T1 Tre nhẾ ng re 89 Mammalian RCC] is N-terminally mefHyÏqfed. 89 N-terminal methylation motif o.cccccccccccccccsssscsseccesecsscessseeveseseseseesensseceseseseesecesseqesneasatseeeseqsetesssesseaseensaseens 94 N-terminal methyltransferase đC[[VỈDJ.
ánh HH TH KH H0 11111101 HH H110 0 rau 98 Anti-d-N-me2/anti-œ-N-me2/3Ser2-RCC1 antibOdÌlớg. ngrong 101 N-terminal methylation facilitates RCC1 interaction with chrOmdfÏH. cccceieeierierree 104 N-terminal methylation on RCC] is necessary for normal THÍÍOSÏS. cv LH HH xe, Ill N-terminal methylation likely facilitates RCC1 bind to DÌNÀ.
re 121 N-terminal tail of RCC1 alone binds chromatin in a N-terminal methylation dependent manner. 130 Constructs And aHfÌPOÏÌð$. HH TH TH HT 01111 HH go 130 Mass spectrometric analysis of RCC] modjficafiOH. các sọc HH 11211212 rrea 13] Tmmunofluorescence THỈCFOSCODD.
Ăn Là HH1 TT HH Ho ko TH TH Tà HỘ kg nh HH. 132 In vitro methylation ASSQV.ecccccsecccssssesessscieveseseiesesesececssnsesssecensaeneseneseucsdedeaeseneneesetecssusaaraneteresetenssberaees 133 Live cell imaging, fluorescence recovery after photobleaching, and fluorescence loss in PHOLODL CACHING oo eececscsssscscesesesenssessssesensscsneseaseasacsassscsaesssesesenseneaensecesseceuenssaeeesaeiauceasaanecaesasaeeteeseaeneeasaaeees 133 CHAPTER IV. GENERAL DISCUSSION 135 JAZ AND DSRNA BINDING PROTEINS 010777. 136 RCC1 AS A CHROMATIN MARKER \.cccssccsssssssscecesseccsssssncesesssesesaccesecsssasesseseseessceesscseeaesacecesssssseeseseeesteasstease 141 N-TERMINAL METHYLATION 2002127.
146 REFERENCE 149 Vill List of Figures FIGURE 1.1 NUCLEAR TRANSPORT SIGNALS.2 SCHEMATIC REPRESENTATION OF THE NUCLEAR PORE COMPLEX. A MODEL OF NUCLEAR IMPORT PATHWAY.1 110110901 vn ng ng ng ng 011012589 16 FIGURE 1.4 A MODEL OF NUCLEAR EXPORT PATHWAY. ong ng HH T4 Hàng 0101 ke 18 FIGURE 1.5 THE GUANINE NUCLEOTIDE CYCLE OF THE RAN OTPASE.90 101 tt H14 1 re23 FIGURE 1.6 A MODEL OF HOW THE RANGTP GRADIENT REGULATES SPINDLE FORMATION, .1 JAZ 1S ANUCLEOCYTOPLASMIC SHUTTLING PROTEIN.- G01 SH HH1 1n g0 1 11c42 FIGURE2.2 EXPORTIN-5 BINDS TO JAZ AND STIMULATES JAZ SHUTTLING IN INTACT CELLS. EXPORTIN-5 IS REQUIRED FOR JAZ SHUTTLING IN INTACT CELLS.4 EXP-5 MEDIATES EXPORT OF JAZ IN DIGITONIN-PERMEABILIZED CELLS.5 JAZ IS A BINDING PARTNER OF ILLE.
Q0 911 1 TH HH ng ng 44 15 T1 KH 1401 14 35 FIGURE2.6 JAZ INTERACTS WITH THE DSRNA BINDING DOMAINS (DSRBDS) OF ILF3.7 JAZ BINDS DIRECTLY TO ILF3, INDEPENDENTLY OF DSRNA.8 JAZ CAN INTERACT WITH ILF3 AND EXP-5 SIMULTANEOUSLY.9 CELLULAR LOCALIZATION OF DIFFERENT TRUNCATIONS OF JÁ Z.10 NUCLEAR IMPORT OF JAZ.ccccccscsscsssssessesseeccesseaesnesseeans secseteseeeee 69 FIGURE 2.11 JAZ IS IMPORTED INTO NUCLEI BY A NON-CLASSICAL PATHWAY. Gà HH HH 1418111 72 FIGURE 3.1 REPRESENTATION OF THE INTERACTION BETWEEN RCC1 AND CHROMATTM.2 RCCI N-TERMINAL METHYLATION. HH TH TH TT TH TT 1304001140 90 FIGURE 3.3 RCC1 N-TERMINAL PHOSPHORYLA TION. con HH H491 1g TH HH0 011031010314 1010101 1x9 92 FIGURE 3.4 IDENTIFICATION OF N-TERMINAL METHYLATION MOTIF.5 IDENTIFICATION OF œ-N-TERMINAL METHYLTRANSFERASE ACTIVITY.6 DETECTION OF ENDOGENOUS RCCI N-TERMINAL METHYLATION.7 METHYLATION OF RCC] REGULATES ITS INTERACTION WITH CHROMOSOMES.8 METHYLATION OF RCC1 REGULATES ITS INTERACTION WITH CHROMOSOMES IN LIVE CELLS.9 METHYLATION OF RCCI IS REQUIRED FOR CORRECT SPINDLE ASSEMBLY AND CHROMOSOME SEGREGATHION.10 ‘TIME-LAPSE IMAGES OF NORMAL AND DEFECTIVE MITOSIS.ccsscsecsssessesceeessaseesessesssesenessescanes 115 TABLE 1, QUANTIFICATION OF MITOTIC DEFECTS IN TRANSFECTED.11 TETHER RCC1 TO CHROMATIN BY FUSION TO HISTONE H2A.12 METHYLATION OF RCC] FACILITATES ITS INTERACTION WITH DNA.13 METHYLATION PROMOTES BINDING OF THE RCC1 TAIL TO MITOTIC CHROMOSOMES IN LIVING UNFIXED MDCK CELLS.
MODEL FOR RCC1 INTERACTION WITH NUCLEOSOME. HH H010 110 n0 ko 128 List of Abbreviations Abbreviation Meaning A Alanine ATP Adenosine 5’-Triphosphate BIB Beta-Like Import Receptor Binding Domain bp Base Pair BSA Bovine-Serum Albumin Cysteine Aspartate Da Dalton DAPI 4’,6-diamidino-2-phenylindole DHCC 3,3’-dihexyloxacarbocyanine DMEM Dulbecco’s Modified Eagle Medium DNA Deoxyribonucleic Acid DTT Dithiothreitol Glutamate EDTA Ethylenediaminetetraacetic Acid Phenylalanine FITC Fluoroscein Isothiocyanate Glycine GDP Guanosince 5’-Diphosphate xi GEF Guanine Nucleotide Exchange Factor GFP Green Fluorescent Protein GGNLS GST-GFP-NLS GlcNAc N-acetylglucosamine GppNHp 5’-Guanylylimidodiphosphate GST Glutathione-S-Transferase GTP Guanosine 5’-Triphosphate H Histidine HEPES N-(2-hydroxyethyl)piperazine-N’-(2- ethanesulfonic Acid) His, Six-Histidine Tag; Sequence: HHHHHH hnRNP Heterogeneuos Nuclear Ribonucleoparticle protein I Isoleucine IgG Immunoglobulin-y IPTG Isopropylthio-B-D-galactoside Lysine kilo- KCl Potassium Chloride hnRNP K Nuclear Shuttling Signal KOAc Potassium Acetate Kerr Dissociation Rate Constant xii KOH Potassium Hydroxide L Leucine M Methionne MDa Megadalton MAPK Mitogen-Activated Protein Kinase MeOH Methanol MEK Mitogen-Activated Protein Kinase Kinase MgCl, Magnesium Chloride MgOAc Magnesium Acetate yg, pl, or ym Microgram, Microliter or Micrometer ml or mg Milliliter or Milligram min Minute MOPS 3-(N-morpholino)propanesulfonic Acid mRNA Messenger RNA | N Asparagine n nano NaCl Sodium Chloride NES Nuclear Export Signal NF-AT Nuclear Factor of Activated T-Cells NLS Nuclear Localization Signal NPC Nuclear Pore Complex NpIC Nucleoplasmin Core Domain xiii NH,-Terminal Domain Nup Nucleoporin Proline PAGE Polyacrylamide Gel Electrophoresis PBS Phosphate-Buffered Saline PFA Paraformaldehyde Isoelectric Point PKA cAMP-Dependent Protein Kinase PKI PKA Inhibitor PMSF Phenylmethylsulfonyl Fluoride Q Glutamine R Arginine Ran Ras-like Nuclear Protein RanBP1, RanBP2, RanBP3 Ran-Binding Protein 1, 2, or 3 RBD Ran-Binding Domain RCCI Regulator of Chromosome Condensation 1 Rabbit Reticulcyte Lysate Ribonucleic Acid Ribonucleoparticle rRNA Ribosomal RNA Serine SDS Sodium Dodecyl Sulfate XIV SV40 Simian Virus 40 Threonine TBE Tris-Borate-EDTA TCA Trichlroroacetic Acid TMRM Tetramethylrhodamine-5-Maleimide TR Texas Red Tris Tris(hydroxymethyl)aminomethane tRNA Transfer RNA TX-100 Triton X-100 Unit Valine Tryptophan WGA Wheat Germ Agglutinin wt Wild-Type Tyrosine IgG-Binding Domain from Protein A Chapter I. General Introduction RanG TP and nuclear transport One of the defining features of a eukaryotic cell is the possession of a nuclear envelope. The nuclear envelope is a continuous double membrane structure that spatially and temporally separates DNA replication and transcription from protein synthesis.