Translational Regulation of mRNA by G-Quadruplex Structures A dissertation submitted to the Department of Chemistry at Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Mark J. Morris August 2012 Dissertation written by Mark Morris B.S., Union College, 2005 Ph.D., Kent State University, 2012 Approved by _______________________________, Chair, Doctoral Dissertation Committee ______________________________ , Advisor, Doctoral Dissertation Committee Soumitra Basu , Ph.D. _______________________________, Member, Doctoral Dissertation Committee William Merrick, Ph.D. _______________________________, Member, Doctoral Dissertation Committee Hanbin Mao, Ph.D. _______________________________, Member, Doctoral Dissertation Committee Roger Gregory, Ph.D. Accepted by _______________________________, Chair, Dept. Chemistry and Biochemistry Michael Tubergen, Ph.D. _______________________________, Dean, College of Arts and Sciences John R.D. Stalvey, Ph.D. ii Table of Contents Page LIST OF FIGURES ……………………………………………………………….……..… vi LIST OF TABLES ………………………………………………………………….……......xii ACKNOWLEDGMENTS …………………………………………………………...………xiii CHAPTER 1 Introduction and Background …………………………………..………… 1 1.1 Discovery of G-quadruplexes ………………………………………………….... 1 1.2 G-quadruplex structures in the 5'-UTR of mRNAs………………………………..5 1.3 Modulation of quadruplex structure and function by small molecules…………….7 CHAPTER 2 An extremely stable G-quadruplex within 5'-UTR of the MT3 matrix metalloproteinase mRNA represses translation in eukaryotic cells...............10 2.1 Introduction ………………………………………………………………………………..10 2.2 Materials and Methods …………………………………………………………………….11 2.3 Results……………………………………………………………………………………….15 2.4 Discussion…………………………………………………………………………………..26 2.5 Conclusion………………………………………………………………………………….30 CHAPTER 3 The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells ………………………………………………………………...31 iii 3.1 Introduction ………………………………………………………………………………...31 3.2 Materials and Methods ………………………………………………………………….....32 3.3 Results and Discussion……………………………………………………………………...36 3.4 Conclusion…………………………………………………………………………………..54 CHAPTER 4 An RNA G-quadruplex is essential for cap-independent translation initiation in human VEGF IRES ………………………………………………………….....55 4.1 Introduction ………………………………………………………………………………..55 4.2 Materials and Methods…………………………………………………………………….57 4.3 Results ………………………………………………………………………………….....62 4.4 Discussion………………………………………………………………………………….77 4.5 Conclusion……………………………………………………………………………..…..82 CHAPTER 5 RNA DOMAIN SWAPPING SHOW CONTEXT DEPENDENT EFFECT OF G-QUADRUPLEXES ON TRANSLATION …………………………………………83 5.1 Introduction ………………………………………………………………………..….….83 5.2 Materials and Methods ……………………………………………………………….…..85 5.3 Results and Discussion…………………………………………………………………….88 5.4 Conclusion……………………………………………………………………………...….95 iv CONCLUDING REMARKS……………………………………………………………...…96 REFERENCES………………………………………………………………………………..97 v List of Figures Page CHAPTER 1 Introduction and Background Figure 1.1. Structure and H-bond formation of a G-tetrad ………………………………..... 1 Figure 1.2. Structure of a G-quadruplex ………………………………………………...….. 2 Figure 1.3 General arrangements of G-quadruplexes based on orientation of the strand……………………………………………………………………………..3 CHAPTER 2 An unusually stable G-quadruplex within 5'-UTR of the MT3 matrix metalloproteinase mRNA represses translation in eukaryotic cell Figure 2.1 Schematic representation of MT3-MMP mRNA …………………………..……11 Figure 2.2 Circular dichroism spectra of M3Q and mut-M3Q in the presence of various concentrations of KCl. …………………………………………………………..16 Figure 2.3 Circular dichroism melting curves of M3Q RNA ……………………………….17 Figure 2.4 Circular dichroism cooling curve and first derivative plot of M3Q RNA………………………………………………………………….…………..18 Figure 2.5 Plot of Tm values for M3Q RNA at various strand concentrations ……..…........20 Figure 2.6 RNase T1 footprinting of M3Q and mut-M3Q. …………………………………21 Figure 2.7 Schematic representation of the plasmids used to investigate the effect of the 5'-UTR of MT3-MMP on translation. …………………….……….…….23 Figure 2.8 Histogram representing the ratio of Renilla/firefly luciferase activities in HeLa cells………………………………………………………………….…23 Figure 2.9 Schematic representation of the plasmids used to investigate the effect of the vi M3Q on translation……………………………………………………………..24 Figure 2.10 Histogram representing the ratio of Renilla/firefly luciferase activities in HeLa cells …………...………………………………………………………...25 Figure 2.11 Circular dichroism first derivative plot of M3Q RNA in the presence of 5 mM KCl…………………………………………………………………..26 CHAPTER 3 The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3- MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells Figure 3.1 CD spectra of 4 µM prefolded (in 100 mM KCl) M3Q in the absence and presence of increasing concentrations of TmPyP4………………………..37 Figure 3.2 CD spectra of TmPyP4 in the absence and presence of folded M3Q………….38 Figure 3.3 Plot of calculated fraction folded vs. TmPyP4 concentration………………….39 Figure 3.4 CD spectra of 4 µM M3Q RNA (in 100 mM KCl) folded in the absence and presence of 0, 2, 5, 10, and 20 µM TmPyP4……………………………...40 Figure 3.5 Native gel shift assay of M3Q (final concentration of about 250 nM) in the absence and presence of increasing concentrations of TmPyP4…………..41 Figure 3.6 NMR spectra of 0.42 mM M3Q titrated with TmPyP4………………………..43 Figure 3.7 Visible absorbtion spectra of TmPyP4 in the absence and presence vii of increasing concentration of prefolded M3Q………………………………...45 Figure 3.8 Visible absorbtion spectra of TmPyP4 in the absence and presence of mut-M3Q……………………………………………………………………46 Figure 3.9 A) Schematic of dual luciferase bi-cistronic constructs. B) Histogram showing % activity of the translation of the Renilla gene as a function of TmPyP4 concentration…………………………………………………..…48 Figure 3.10 Histogram representing the ratio of Renilla to firefly mRNA CT values in HeLa cells determined by qRT-PCR………………………………50 Figure 3.11 CD-melting spectrum of the DNA version of M3Q (4 µM) in the presence of 100 mM KCl…………………………………………………….52 Figure 3.12 Plot of fraction folded vs. TmPyP4 concentration of 4 µM prefolded (in 100 mM KCl) DNA version of M3Q in the absence and presence of increasing concentrations of TmPyP4……………………...53 CHAPTER 4 An RNA G-quadruplex is essential for cap-independent translation initiation in human VEGF IRES Figure 4.1. Primary nucleotide sequence of the human VEGF IRES-A ……....................56 Figure 4.2. RNase T1 footprinting in the presence of 150 mM K+, 150 mM Li+ and 1 mM MgCl2 …………………………………………………………………………62 viii Figure 4.3 DMS footprinting in the presence of 150 mM K+ or 150 mM Li+……………………………………………………………………………..63 Figure 4.4 Schematic of a subset of G-quadruplex structures that shows the different G- stretches……………………………………………………………………….64 Figure 4.5 Circular dichroism (CD) spectra of an oligoribonucleotide encompassing the protected region in hVEGF IRES A…………………………………………..66 Figure 4.6 Schematic of various dual luciferase bi-cistronic constructs………………....68 Figure 4.7 Histogram showing % activity of the mutant constructs normalized to the wild type construct……………………………………………………..69 Figure 4.8 Histogram representing the ratio of Renilla to firefly luciferase activities in HeLa cells……………………………………………………...…70 Figure 4.9 Scanned images of gels showing RNase T1 footprinting of the 293 nt human VEGF IRES-A and its various mutants……………………….71 Figure 4.10 Scanned images of gels showing DMS footprinting of the 293 nt human VEGF IRES-A and its various mutants………………..…….73 Figure 4.11 Histogram showing % activity of the dual luciferase rescue mutant construct (Rescue Quad) normalized to its parent construct (G774, 789U)………………………………………………………76 ix Figure 4.12 Scanned image of a gel showing RNase T1 footprinting of the mutant Rescue Quad version of the transcribable 293 nt human VEGF IRES-A in the presence of K+ and Li+………………..77 Figure 4.13 Histograms representing quantitation of the gel shown in Figure 4.12………………………………………………………………..79 CHAPTER 5 Effect of G-quadruplex domain exchange on translation Figure 5.1 Schematic of RNA G-quadruplex swapping………………...……………….83 Figure 5.2 Dual luciferase reporter assay results demonstrating the roles in translation of endogenous VEGF G-quadruplex, the 4MVF sequence, the M3Q G-quadruplex forming sequence , and the NRAS quadruplex forming sequence all within the 5' UTR of hVEGF IRES-A…………………………………………………………...89 Figure 5.3 Scanned image of a gel showing results of RNase T1 footprinting of the wild-type, quadrupole mutant, M3Q, and the NRAS quadruplex forming sequence inserted in the 5'-UTR of the hVEGF IRES-A………………………………………………………………92 Figure 5.4 Dual luciferase reporter assay results demonstrating the roles x in translation of endogenous M3Q G-quadruplex, the mutated sequence and the QVEG G-quadruplex forming sequence all within the 5' UTR of MT3-MMP…………………………………………….94 xi List of Tables Page CHAPTER 2 An unusually stable G-quadruplex within 5'-UTR of the MT3 matrix metalloproteinase mRNA represses translation in eukaryotic cell Table 2.1 Tm values for M3Q RNA in the presence of various cations………………………19 Table 2.2 Thermodynamic parameters for the folding of M3Q……………………………....20 CHAPTER 5 Effect of G-quadruplex domain exchange on translation Table 5.1 Primer sequences used for various RNA G-quadruplex