STRUCTURAL AND FUNCTIONAL ANALYSIS OF CRITICAL PROTEINS INVOLVED IN mRNA DECAY CHENG ZHIHONG NATIONAL UNIVERSITY OF SINGAPORE 2006 STRUCTURAL AND FUNCTIONAL ANALYSIS OF CRITICAL PROTEINS INVOLVED IN mRNA DECAY CHENG ZHIHONG (B.Sc) Ease China University of Science and Technology A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 To My Wife i Index Index………………………………………………………………………………………i Acknowledgements …………………………………………………………….……… ii Table of Contents……………………………………………………………………… iii Abstract …………………………………………………………………………… viii Lists of Figures…………………………………………………………………… .… x Lists of Tables ……………………………………………………………………….…xii Lists of Abbreviations ……………………………………………………………… .xiii References………………………………………………………………………… .…147 Appendix I MOPS Minimal Medium …………………………………………… 168 Appendix II Publication list…………………………………………………………170 ii Acknowledgements I would like to thank sincerely my supervisor, Dr Song Haiwei, for offering me an opportunity being a postgraduate. Without his great guidance, patience and advice, I could not finish my Ph.D program successfully. Many thanks also go to our collaborator from Howard Hughes Medical Institute, The University of Arizona, Professor Roy Parker, Drs. Jeff Coller and Denise Muhlrad, for their great experiment data and precious discussions. I would like to give my appreciation to the members of my thesis committee: Associate Professor Kunchithapadam Swaminathan, Associate Professor Wang Yue from Institute of Molecular and Cell (IMCB) and Assistant Professor J Sivaraman from Department of Biological Sciences (NUS), for guidance and discussions throughout my studies. I also thank all my colleagues in my laboratory for their kind help. Thanks especially go to Dr. Kong Chunguang, Dr. Wu Mousheng, Miss She Meipei, Miss Chen Nan and Dr Zhou Zhihong for all the valuable discussions in all the experiments. I want to thank Dr. Christian, Dr. Rohini, Miss Portia and Sharon, for their critical reading of the manuscript of this thesis, and Lim Mengkiat for his great help in the experiment. I also thank all administrative staffs in Department of Biological Sciences (NUS) and IMCB for their supports. Thanks also go to the shared facilities in IMCB including DNA sequencing facility and Mass Spectrometry facility for experimental supports. Finally, I would like to thank my family, especially my wife for her full support, patience, encouragement and inspiration all the time. I could not image the situation without her precious support. iii Table of contents Chapter Introduction 1.1. Biological significance of mRNA decay……………………………………… 1.2. General mRNA decay pathway…………………………………………………1 1.2.1. Deadenylation…………………………………………………………… 1.2.2. Decapping……………………………………………………………… 1.2.2.1. The Dcp1-Dcp2 Decapping enzyme complex………………………6 1.2.2.2. Regulation of the decapping activity…………………………… 1.2.2.3. Scavenger decapping enzyme DcpS……………………………… .9 1.2.3. Enzymes involved in mRNA body degradation…………………………10 1.2.3.1. 5’ to 3’ degradation by Xrn1……………………………………….10 1.2.3.2. 3’ to 5’ degradation by the exosome complex………………… 11 1.3. mRNA quality control mechanisms targeting aberrant mRNAs………………13 1.3.1. Nonsense-mediated mRNA decay…………………………………… .13 1.3.1.1. NMD factors……………………………………………………….15 1.3.1.2. Translation and NMD…………………………………………… .16 1.3.1.3. Definition of premature termination codons……………………….18 1.3.1.4. Recognition of PTC in mammals……………………………… 20 1.3.2. Nonstop mRNA decay ………………………………………………… 22 1.4. Specialized mRNA decay pathways………………………………………… .23 1.5. Project I: Structural and functional studies of Ski8………………………… .27 1.5.1. Brief introduction of Ski8……………………………………………… 27 iv 1.5.2. Structural characteristics of WD-repeat proteins……………………… .29 1.5.3. Aims of this project………………………………………………………33 1.6. Project II: Structural and functional studies of Dhh1…………………………34 1.6.1. Brief review of Dhh1…………………………………………………….34 1.6.2. Structural characterization of Superfamily helicases…………… .35 1.6.3. Aims of this project………………………………………………………40 1.7. Project III: Structural and functional analysis of hUpf1……………………….41 1.7.1. Previous functional and biochemical studies of Upf1……………… .41 1.7.2. Structural studies of Superfamily helicases……………………………43 1.7.3. Aims of this project………………………………………………………44 Chapter Cloning, Protein Purification, Crystallization and Structure Determination 2.1. Gene cloning and protein expression strain construction…………………… .45 2.1.1. Yeast genomic DNA isolation………………………………………… .45 2.1.2. Polymerase chain reaction (PCR) ……………………………………….45 2.1.3. Agarose gel electrophoresis…………………………………………… .46 2.1.4. Purification of PCR products…………………………………………….46 2.1.5. Enzyme digestion, dephosphorylation and purification…………… .46 2.1.6. Ligation and transformation………………………………………… .47 2.1.7. Plasmid preparation and positive clone screening……………………….47 2.1.8. DNA sequencing……………………………………………………… 47 2.1.9. E. coli expression strain transfomation ………………………………….48 2.1.10. Protein expression test and expression strain storage……………………48 2.1.11. SDS-PAGE………………………………………………………………48 v 2.2. Protein purification………………………………………………………… .49 2.2.1. Large-scale cell culture for protein expression………………………… 49 2.2.2. Purification procedures……………………………………………… 49 2.2.2.1. Cell lysis………………………………………………………… .51 2.2.2.2. First GST affinity column chromatography……………………….51 2.2.2.3. Second GST affinity column chromatography…………………….51 2.2.2.4. Ion exchange chromatography………………………………… 51 2.2.2.5. Gel filtration chromatography………………………………… .52 2.3. Crystallization……………………………………………………………… .53 2.4. Structure determination…………………………………………………… .55 2.4.1. Heavy atom derivative preparation………………………………… 55 2.4.2. Data collection and processing………………………………………… 56 2.4.2.1. Data collection and processing of SeMet Ski8…………………….56 2.4.2.2. Data collection and processing of the Br derivative of Dhh1…… 57 2.4.2.3. Data collection and processing of SeMet hUpf1……………… 58 2.4.3. Structure determination………………………………………………… 59 2.4.3.1. Phasing, modeling and refinement of Ski8…………………… .59 2.4.3.2. Phasing, modeling and refinement of Dhh1……………………….62 2.4.3.3. Structure determination of hUpf1………………………………….65 2.5. Biochemical and molecular biological experiments……………………… .72 2.5.1. Experiments for Ski8………………………………………………… .72 2.5.1.1. Site-directed mutagenesis and yeast two-hybrid assay……………72 2.5.1.2. GST pull-down assay…………………………………………… .72 vi 2.5.2. Experiments for Dhh1……………………………………………………73 2.5.2.1. CD-spectroscopy………………………………………………… .73 2.5.2.2. Mutagenesis and in vivo mRNA turnover assays………………….73 2.5.2.3. Limited Proteolysis……………………………………………… .74 2.5.2.4. In vitro RNA binding assay……………………………………… 74 2.5.3. Experiments for hUpf1………………………………………………… 75 2.5.3.1. Mutagenesis and In vitro ATPase activity…………………………75 2.5.3.2 In vitro ATP binding assay……………………………………… .75 2.5.3.3. In vitro RNA binding assay……………………………………… 76 2.5.3.4. In vivo NMD analysis and P-body formation………………… .76 2.5.3.5. Surface plasmon resonance (SPR) ……………………………… .77 Chapter Crystal Structure and Mutagenesis Studies of Ski8 3.1. Results…………………………………………………………………………78 3.1.1. Overall structure determination………………………………………….78 3.1.2. Comparison with other WD repeat proteins…………………………… 80 3.1.3. Location of protein-protein interaction sites on the β propeller…………83 3.1.4. Mutational Analysis of Ski8…………………………………………… 87 3.2. Discussion………………………………………………………………… ….90 Chapter Structural and Functional Analysis of Dhh1 4.1. Results ……………………………………………………………………… 94 4.1.1. Structural overview of the Dhh1…………………………………………94 4.1.2. Structural Comparison ……………………………………………… 96 4.1.3. Location of the conserved sequence motifs…………………………… .98 vii 4.1.4. Interactions of the conserved Motifs……………………………………103 4.1.5. Identification of residues required for RNA binding ………………… 107 4.1.6. Conformational changes in Dhh1………………………………………114 4.2. Discussion………… ……………………………………………………… 116 Chapter Structural and Functional Insights into hUpf1 5.1. Results……………………………………………………………………… 120 5.1.1. Overall structure ……… ………………………………………………120 5.1.2. Nucleotide Binding site and ATP hydrolysis………………………… .124 5.1.3. Conformational change during the Upf1 ATPase cycle……………… 128 5.1.4. Allosteric effect of ATP binding coupled with RNA binding .……… .133 5.1.5. Differential effects of Upf1 mutants on P-body formation…………… 141 5.2. Discussion………………………………………………………………… .143 158 Lohman,T.M., Chao,K., Green,J.M., Sage,S., and Runyon,G.T. (1989). Large-scale purification and characterization of the Escherichia coli rep gene product. J. Biol. Chem., 264, 10139-10147. Lorentzen,E. and Conti,E. (2005). 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Reagents Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Phe Pro Ser Thr Trp Tyr Val Adenine Guanine Cytosine Uracil Thymine Concentration (mM) 2.5 2 0.5 3 4 2 18.3 0.5 1 1 Quantity (mg) 356.4 526.75 264.2 266.2 60.6 441.3 438.3 300.4 155.2 524.8 1049.6 730.3 330.4 230.2 1922.6 238.2 102.1 362.4 703.2 171.6 151.1 111.1 112.1 126.1 169 (3) 5000x Micronutrients (500ml) Reagents Na2MoO4•2H2O H3BO3 CoCl2 CuSO4 MnCl2 Zn(OAc)2 (4) Quantity (gram) 0.463 0.618 0.178 0.043 0.396 0.072 2000x Vitamin mix (10ml): Add a little NaOH to improve the solubility, sterilize with 0.2μm filter. Reagents Thiamine Pantothenic acid p-hydroxybenzoic acid p-aminobenzoic acid 2,3-dihydroxybenzoic acid Quantity (gram) 67.4 47.6 32 27.4 30.8 The recipe of MOPS minimal medium (10 liter) Reagents or buffers 10x MOPS buffer 20% Glucose 5x Amino acid stock Water 0.132 M K2HPO4 (be added after water) 2000x Vitamin stock 5000x Micronutrients Volumn (ml) 1000 300 2000 6600 100 0.2 170 Appendix II Publication list 1) Cheng Z, Liu Y, Wang C, Parker R, Song H. Crystal structure of Ski8p, a WDrepeat protein with dual roles in mRNA metabolism and meiotic recombination. Protein Sci. 2004 Oct;13(10):2673-84. (Cover story) 2) Cheng Z, Coller J, Parker R, Song H. Crystal structure and functional analysis of DEAD-box protein Dhh1p. RNA. 2005 Aug;11(8):1258-70. 3) Cheng Z, Muhlrad D, Lim M, Parker R, Song H. Structural and functional insights into the human Upf1 helicase core. EMBO. J 2007 Jan 10;26(1):253-64. 171 172 173 [...]... control of mRNA translation and degradation is important for gene expression in eukaryotic cells mRNA decay, including mRNA quality control, is a multistep processing event composed of deadenylation, decapping and degradation of the mRNA body Three proteins, hUpf1, Dhh1 and Ski8 involved in eukaryotic mRNA decay were structurally and functionally studied in this thesis Ski8 is a WD-repeat protein with... Upf3b) and is also one of the components of the exon-exon junction complex (EJC; Le Hir et al., 2000; Kim et al., 2001) Upf2 is perinuclear and might attach to exporting mRNAs by interacting with Upf3 (Lykke-Andersen et al., 2000) The crystal structure of the interacting domains of hUpf2 and hUpf3b shows that the RNA-binding domain (RBD) of Upf3 is involved in the interaction with one MIF4G domain of Upf2,... potential mapping combined with mutagenesis reveals that motifs I, V, and VI are involved in RNA binding In addition, trypsin digestion of the truncated Dhh1 in the absence or presence of RNA or ligand suggests that ATP binding enhances an RNAinduced conformational change Interestingly, some mutations located in the conserved motifs and at the interface between the two RecA-like domains confer dominant negative... blocks mRNA decapping Stimulate mRNA decapping Lsm7, eIF4G, eIF4A, eIF4B, Pab1 Dcp1, Dcp2, Edc1, Edc2, Small basic proteins Edc3 Contains five Edc3 regulates decapping of conserved domains RPS28B mRNA Pat1 No recognizable motifs Lsm1-7 Sm-like proteins Dhh1 DEAD-box protein Stimulates mRNA decapping and associates with deadenylases Stimulates mRNA decapping and formation of P-bodies in vivo Stimulate mRNA. .. Negative regulators include the poly(A)-binding protein 1 (Pab1) and the cap-binding protein (eIF4E) Pab1 couples decapping with deadenylation and inhibits decapping by promoting the formation of the translation initiation complex (Caponigro et al., 1995; Morrissey et al., 1999) In vivo and in vitro assays have shown that eIF4E can inhibit decapping activity by competitively binding to the 5’-cap structure... ssRNA binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis These conformational changes alter the likely ssRNA-binding channel in a manner that can explain how ATP binding destabilizes ssRNA binding to Upf1 Keywords: mRNA decay, nonsense-mediated mRNA decay, WD-repeat protein, Ski8, DEAD-box protein, Dhh1, RNA helicase, Upf1, X-ray crystallography x Lists of Figures... of hUpf1hd upon nucleotide binding and hydrolysis………………………………………………………… 132 Figure 5-5 Protein gel filtration profile of hUpf1 WT and mutants 1B∆ and 1C∆……………………………………………………………… 133 Figure 5-6 The channel between domains 1B and 1C involved in ssRNA binding………………………………………………………………….135 Figure 5-7 Allosteric effects of ATP binding/hydrolysis on RNA binding……… 137 Figure 5-8 Surface plasma resonance analysis. .. The crystal structure of the N-terminal domain of SMG7 reveals that this protein contains a 14-3-3-like phosphoserine-binding domain, which is involved in the association with phosphorylated Upf1 (Fukuhara et al., 2005) Conservation of this 14-3-3-like domain among SMG5, SMG6 and SMG7 suggests that these proteins act as similar adaptors in mediating dephosphorylation of Upf1 in NMD Name Yeast Mammal... eRF1 in a similar way as its prokaryotic counterpart RF3 (Frolova et al., 1996; Zavialov et al., 18 2001) Sequence analysis indicates that eRF3 contains at least two functional regions: an N-terminal region, which is not necessary for translation termination but is involved in binding to poly(A)-binding protein, suggesting a link between the termination event and the initiation process in protein biosynthesis,... resonance analysis of RNA binding to hUpf1…………139 Figure 5-9 Structure comparison of hUpf1hd-AMPPNP with hUpf1hd-ATPγS……………………………………………………… 141 Figure 5-10 Visualization of Dcp2-GFP in live yeast strains expressing Upf1 mutants………………………………………………………… 143 xii Lists of Tables Table 1-1 Critical proteins involved in general mRNA decay pathway…………… 3 Table 1-2 Regulators of mRNA decapping……………………………………… . STRUCTURAL AND FUNCTIONAL ANALYSIS OF CRITICAL PROTEINS INVOLVED IN mRNA DECAY CHENG ZHIHONG NATIONAL UNIVERSITY OF SINGAPORE 2006 STRUCTURAL AND FUNCTIONAL ANALYSIS. ANALYSIS OF CRITICAL PROTEINS INVOLVED IN mRNA DECAY CHENG ZHIHONG (B.Sc) Ease China University of Science and Technology A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. expression in eukaryotic cells. mRNA decay, including mRNA quality control, is a multi- step processing event composed of deadenylation, decapping and degradation of the mRNA body. Three proteins,