Project

Back to overview

Metal Ion-Guided Assembly and Structures of the Catalytic Core of Ribozymes

Applicant Sigel Roland K. O.
Number 143750
Funding scheme Project funding (Div. I-III)
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Inorganic Chemistry
Start/End 01.10.2012 - 31.03.2016
Approved amount 469'017.00
Show all

Keywords (6)

NMR; Coordination Chemistry; Metal Ions; Mammalian Ribozyme; Splicing; Group II Intron Ribozyme

Lay Summary (English)

Lead
Lay summary

Metal ions are necessary for folding and function of catalytic RNA molecules. However, the structural and mechanistic roles of these ions are still largely in the dark. Na(I) and Mg(II) are usually assumed to be the sole metallic cofactors involved with nucleic acids in living systems, both being most abundant and freely available in the cell. The recognition of these and other metal ions by nucleic acids is poorly understood as is their effect on structure, folding, and catalytic activity.

This study focuses on the metal ion assisted assembly of the catalytic core of two different ribozymes, i.e. a group II intron from yeast as well as a mammalian ribozyme, and the thermodynamic and structural characterization of these interactions. A multidisciplinary approach is applied by a combination of tools from Coordination and Analytical Chemistry as well as Structural Biology, mainly biomolecular NMR spectroscopy. Our results will not only contribute to the understanding of the metal ion-promoted structure, folding, and mechanism of the RNAs catalytic core, but also promise to have a significant impact on the Biological Inorganic Chemistry of RNAs and on RNA Biochemistry in general.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Metal ion binding to an RNA internal loop: An NMR Study
Bartova Simona, Alberti Elena, Sigel Roland K. O., Donghi Daniela (2016), Metal ion binding to an RNA internal loop: An NMR Study, in Inorganica Chimica Acta, 452, 104-110.
Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme
Skilandat Miriam, Rowinska-Zyrek Magdalena, Sigel Roland K .O. (2016), Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme, in RNA, 22(5), 750-763.
Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR
Bartova Simona, Pechlaner Maria, Donghi Daniela, Sigel Roland K. O. (2016), Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR, in JBIC Journal of Biological Inorganic Chemistry, 21(3), 319-328.
Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns
Kowerko Danny, König Sebastian L. B., Skilandat Miriam, Kruschel Daniela, Hadzic Mélodie C. A. S., Cardo Lucia, Sigel Roland K. O. (2015), Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns, in Proceedings of the National Academy of Sciences, 112(11), 3403-3408.
Explicit analytic equations for multimolecular thermal melting curves
Böttcher Albrecht, Kowerko Danny, Sigel Roland K.O. (2015), Explicit analytic equations for multimolecular thermal melting curves, in Biophysical Chemistry, 202, 32-39.
Protonation-Dependent Base Flipping at Neutral pH in the Catalytic Triad of a Self-Splicing Bacterial Group II Intron
Pechlaner Maria, Donghi Daniela, Zelenay Veronika, Sigel Roland K. O. (2015), Protonation-Dependent Base Flipping at Neutral pH in the Catalytic Triad of a Self-Splicing Bacterial Group II Intron, in Angewandte Chemie, 127(33), 9823-9826.
NMR structure of the 5' splice site in the group IIB intron Sc.ai5 --conformational requirements for exon-intron recognition
Kruschel Daniela, Skilandat Miriam, Sigel Roland K. O. (2014), NMR structure of the 5' splice site in the group IIB intron Sc.ai5 --conformational requirements for exon-intron recognition, in RNA, 20(3), 295-307.
Solution structure and metal ion binding sites of the human CPEB3 ribozyme’s P4 domain
Skilandat Miriam, Rowinska-Zyrek Magdalena, Sigel Roland K. O. (2014), Solution structure and metal ion binding sites of the human CPEB3 ribozyme’s P4 domain, in JBIC Journal of Biological Inorganic Chemistry, 19(6), 903-912.
Structure determination of noncanonical RNA motifs guided by 1H NMR chemical shifts
Sripakdeevong Parin, Cevec Mirko, Chang Andrew T., Erat Michèle C., Ziegeler-Koschinat Melanie, Zhao Qin, Fox George E., Gao Xiaolian, Kennedy Scott D., Kierzek Ryszard, Nikonowicz Edward P., Schwalbe Harald, Sigel Roland K. O., Turner Douglas H., Das Rhiju (2014), Structure determination of noncanonical RNA motifs guided by 1H NMR chemical shifts, in Nature Methods, 11(4), 413-416.
The Role of Magnesium(II) for DNA Cleavage Site Recognition in Group II Intron Ribozymes - Solution Structure and Metal Ion Binding Sites of the RNA·DNA Complex
Skilandat Miriam, Sigel Roland K. O. (2014), The Role of Magnesium(II) for DNA Cleavage Site Recognition in Group II Intron Ribozymes - Solution Structure and Metal Ion Binding Sites of the RNA·DNA Complex, in Journal of Biological Chemistry, 289(30), 20650-20663.
A QM/MM refinement of an experimental DNA structure with metal-mediated base pairs
Kumbhar Sadhana, Johannsen Silke, Sigel Roland K. O., Waller Mark P., Müller Jens (2013), A QM/MM refinement of an experimental DNA structure with metal-mediated base pairs, in Journal of Inorganic Biochemistry, 127, 203-210.
Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids
Sigel Roland K. O., Skilandat Miriam, Sigel Astrid, Operschall Bert P., Sigel Helmut (2013), Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids, in Sigel Astrid (ed.), Springer Netherlands, Dordrecht, 191-274.
Hexaamminecobalt(III) - Probing Metal Ion Binding Sites in Nucleic Acids by NMR Spectroscopy
Rowinska-Zyrek Magdalena, Skilandat Miriam, Sigel Roland K. O. (2013), Hexaamminecobalt(III) - Probing Metal Ion Binding Sites in Nucleic Acids by NMR Spectroscopy, in Zeitschrift für anorganische und allgemeine Chemie, 639(8-9), 1313-1320.
Intrinsic Acid-Base Properties of a Hexa-2′-deoxynucleoside Pentaphosphate, d(ApGpGpCpCpT): Neighboring Effects and Isomeric Equilibria
Domínguez-Martín Alicia, Johannsen Silke, Sigel Astrid, Operschall Bert P., Song Bin, Sigel Helmut, Okruszek Andrzej, González-Pérez Josefa María, Niclós-Gutiérrez Juan, Sigel Roland K. O. (2013), Intrinsic Acid-Base Properties of a Hexa-2′-deoxynucleoside Pentaphosphate, d(ApGpGpCpCpT): Neighboring Effects and Isomeric Equilibria, in Chemistry - A European Journal, 19(25), 8163-8181.
Structure and Conformational Dynamics of the Domain 5 RNA Hairpin of a Bacterial Group II Intron Revealed by Solution Nuclear Magnetic Resonance and Molecular Dynamics Simulations
Pechlaner Maria, Sigel Roland K. O., van Gunsteren Wilfred F., Dolenc Jožica (2013), Structure and Conformational Dynamics of the Domain 5 RNA Hairpin of a Bacterial Group II Intron Revealed by Solution Nuclear Magnetic Resonance and Molecular Dynamics Simulations, in Biochemistry, 52(40), 7099-7113.
NMR Spectroscopy in Bioinorganic Chemistry
Donghi Daniela, Johannsen Silke, Sigel Roland K. O., Freisinger Eva (2012), NMR Spectroscopy in Bioinorganic Chemistry, in CHIMIA International Journal for Chemistry, 66(10), 791-797.
The structural stabilization of the   three-way junction by Mg(II) represents the first step in the folding of a group II intron
Donghi Daniela, Pechlaner Maria, Finazzo Cinzia, Knobloch Bernd, Sigel Roland K. O. (2012), The structural stabilization of the   three-way junction by Mg(II) represents the first step in the folding of a group II intron, in Nucleic Acids Research, 41(4), 2489-2504.

Collaboration

Group / person Country
Types of collaboration
CERM, Florence Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
PD Dr. Eva Freisinger, Universität Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Meitian Wan & Dr. Vincent Olieric, PSI Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Dr. Helena Kovacs, Bruker Biospin, Fällanden (ZH) Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Symposium Functional Nucleic Acids: Chemistry, Biology, and Materials Applications, PacifiChem 2015 Talk given at a conference Structure and Metal Ion Binding Properties of the Mammalian CPEB3 Ribozyme 15.12.2015 Honolulu, United States of America Sigel Roland K. O.;
Fall Meeting of the Swiss Chemical Society Poster Characterization of Mg2+ binding sites in the CPEB3 ribozyme studies by NMR spectroscopy 04.09.2015 Lausanne, Switzerland Skilandat Miriam; Kenneth Adea; Sigel Roland K. O.;
17th International Conference on Biological Inorganic Chemistry (ICBIC17) Talk given at a conference RNA-RNA Interactons on the Single Molecule Level: Heterogeneity Induced by Metal Ions and the Effect of a Crowded Environment 20.07.2015 Peking, China Sigel Roland K. O.;
International Symposium on Metal Complexes 2015 (ISMEC) Talk given at a conference Towards understanding the folding dynamics of the G-quadruplex sequence at the 5’-UTR of the human BCL2 mRNA 24.06.2015 Wroclaw, Poland Sigel Roland K. O.; Domínguez-Martín Alicia;
International Symposium on Metal Complexes 2015 (ISMEC) Talk given at a conference How H+ and Mg2+ shape and form complex RNA structures 24.06.2015 Wroclaw, Poland Sigel Roland K. O.;
PostDocDay Talk given at a conference Dynamics and stability of the biologically relevant BCL2 RNA G-quadruplex structure 21.05.2015 Au-Wädenswil, Switzerland Domínguez-Martín Alicia; Sigel Roland K. O.;
5th International Meeting on G-quaduplex Nucleic Acids Poster Gene regulation by RNA G-quadruplexes. New insights into the Gquadruplex structure at the 5'UTR of the BCL2 mRNA human protooncogene 04.05.2015 Bordeaux, France Domínguez-Martín Alicia; Sigel Roland K. O.;
Fall Meeting of the Swiss Chemical Society Poster Structural determination of the core region of the group II intron Sc.ai5γ and the role of the divalent metal ions in folding and structure 11.09.2014 Zürich, Switzerland Sigel Roland K. O.; Gashi Serranda;
Fall Meeting of the Swiss Chemical Society Poster New insights into the folding and NMR structure of the human RNA BCL2 G-quadruplex 11.09.2014 Zürichz, Switzerland Sigel Roland K. O.; Domínguez-Martín Alicia;
12th European Biological Inorganic Chemistry Conference Poster Structure ans stability of the human BLC2 RNA G-quadruplex 24.08.2014 Zürich, Switzerland Domínguez-Martín Alicia; Sigel Roland K. O.;
12th European Biological Inorganic Chemistry Conference Poster Structural investigation of the D1κζext region of group II intron Sc.ai5γ 24.08.2014 Zürich, Switzerland Pechlaner Maria; Sigel Roland K. O.; Gashi Serranda;
12th European Biological Inorganic Chemistry Conference Poster The CPEB3 ribozyme pseudoknot is tied up by magnesium(II) 24.08.2014 Zürich, Switzerland Skilandat Miriam; Sigel Roland K. O.;
2nd Symposium on Functional Metal Complexes that Bind to Biomolecules, COST Action CM1105 Talk given at a conference Potassium dependent folding and structure of the human BCL2 RNA G-quadruplex 22.08.2014 Zürich, Switzerland Sigel Roland K. O.; Domínguez-Martín Alicia;
Euromar2014 – Magnetic Resonance Conference Poster NMR structural studies on the human BLC2 RNA G-quadruplex froming sequence 29.06.2014 Zürich, Switzerland Sigel Roland K. O.; Domínguez-Martín Alicia;
GDR G-Quadruplex Meeting 2014 Poster Understanding the Stability and Structure of the Human BCL2 RNA G-Quadruplex Sequence 20.03.2014 Grenoble, France Sigel Roland K. O.; Domínguez-Martín Alicia;
Fall Meeting of the Swiss Chemical Society Poster First insight into the structure and metal-ion binding sites of the human CPEB3 ribozyme 06.09.2013 Lausanne, Switzerland Sigel Roland K. O.; Skilandat Miriam;
International Conference on Biological Inorganic Chemistry (ICBIC 16) Talk given at a conference The Irving-Williams Series on the Single Molecule Level: Controlling Strengths and Kinetics of RNA-RNA Interactions 22.07.2013 Grenoble, France Sigel Roland K. O.;
18th Annual Meeting of the RNA Society Poster Metal ions determine the kinetics and thermodynamics of single RNA-RNA associations according to the Irving-Williams series 11.06.2013 Davos, Switzerland Sigel Roland K. O.;
18th Annual Meeting of the RNA Society Poster The influence of metal-ions on the structure of the CPEB3 ribozyme's P4 region 11.06.2013 Davos, Hungary Sigel Roland K. O.; Skilandat Miriam;
3rd Annual User group Meeting of BioNMR Poster Di- and trivalent metal ions stabilize the folding control element of group II introns 10.06.2013 Budapest, Hungary Sigel Roland K. O.; Pechlaner Maria;
ZiNG Bioinorganic Chemistry Conference Talk given at a conference MINAS – A Quest for Common and Uncommon Metal Coordination Patterns in Nucleic Acids 19.02.2013 Lanzarote, Spain Sigel Roland K. O.;
6th Asian Conference on Biological Inorganic Chemistry (AsBIC-6) Talk given at a conference How Metal Ions Direct the First Steps of Folding of a Large Ribozyme 05.11.2012 Hong Kong, China Sigel Roland K. O.;


Self-organised

Title Date Place
12th European Biological Inorganic Chemistry Conference 24.08.2014 Zürich, Switzerland
2nd International Symposium on Functional Metal Complexes that Bind to Biomolecules, COST Action CM1105 22.08.2014 Zürich, Switzerland

Awards

Title Year
Forschungskredit der Universität Zürich 2015
Forschungskredit der Universität Zürich 2014
Fellowship der Fundación Ramón Areces 2013

Associated projects

Number Title Start Funding scheme
192153 Principles of Metal-Ion-Assisted Folding and Structure of Functional RNAs 01.04.2020 Project funding (Div. I-III)
165868 Metal Ions in Structure and Function of Regulatory RNAs 01.04.2016 Project funding (Div. I-III)
124834 Coordination Chemistry within the Core of Large RNAs: Regulating Tertiary Contacts and Function 01.04.2009 Project funding (Div. I-III)

Abstract

1.SUMMARY AND CENTRAL AIMS OF THE STUDYMetal ions are necessary for folding and function of catalytic RNA molecules. However, the structural and mechanistic roles of these ions are still largely in the dark. Na+ and Mg2+ are usually assumed to be the sole metallic cofactors involved with nucleic acids in living systems, both being most abundant and freely available in the cell. The recognition of these and other metal ions by nucleic acids is poorly understood as is their effect on structure, folding, and catalytic activity. This study focuses on the metal ion assisted assembly of the catalytic core of two different ribozymes, i.e., group II introns and the mammalian CPEB3 ribozyme, and the thermodynamic and structural characterization of these interactions. A multidisciplinary approach is applied by a combination of tools from Coordination and Analytical Chemistry as well as Structural Biology. Our results will not only contribute to the understanding of the metal ion-promoted structure, folding, and mechanism of the RNAs catalytic core, but also promise to have a significant impact on the Biological Inorganic Chemistry of RNAs and on RNA Biochemistry in general. AIM A: Understanding the Assembly of the Group II Intron Catalytic CoreGroup II introns are among the largest occurring RNAs in Nature and are closely related to the eukaryotic spliceosomal machinery. These self-splicing introns exhibit a straight folding pathway devoid of kinetic traps. Folding is initiated by Mg2+ coordination, whereby the kappa-zeta region within domain 1 (D1) is the key element for the first phase of folding. The last step of folding is the Mg2+-assisted docking of domain 5 (D5). We have just solved the solution structure of this central kappa-zeta region and characterized its structural change upon Mg2+ binding. It turned out that an additional extended helix of kappa-zeta is needed for D5 docking: Here we concentrate on the folding, structure and rearrangement of this extended kappa-zeta region. We will first investigate the Mg2+ requirement and structural changes within this three-way junction. Second, the Mg2+-dependent association of the extended kappa-zeta RNA with D5, the specific Mg2+ coordination, as well as the structure of the about 100 nucleotide long extended kappa-zeta/D5 complex will be characterized and solved by NMR. This RNA is unique and perfectly suited for bioinorganic studies because of its high affinity for Mg2+ and the associated structural change. In addition, it also represents a minimal catalytic core of group II introns.AIM B: Folding, Structure, and Mechanism of the Mammalian CPEB3 RibozymeThe CPEB3 ribozyme is an intronic sequence within the cytoplasmatic polyadenylation element-binding protein 3. This RNA belongs to the class of hepatitis delta virus (HDV)-like self-cleaving ribozymes and is special because it is one of two ribozymes occurring in mammals. The human homologue is polymorphic in sequence whereby the nature of one specific nucleotide is linked to cleavage activity and the performance of episodic memory tasks. Like all RNAs, also structure and function of HDV-like ribozymes are strictly dependent on metal ions. Here we will concentrate on the elucidation and characterization of the interplay between different mono- and divalent metal ions and the polymorphs of the ribozyme with regard to folding, structure and mechanism. X-ray crystallography and NMR will be mainly applied to elucidate the structure, determine the nature of metal ion-binding pockets, as well as their role in folding and catalytic core formation. Our ultimate goal is to understand the coordination-chemical basis of the accelerating or inhibiting influence of metal ions in a motion-structure-function relationship of this unique mammalian ribozyme.AIM C: Metal Ion Coordination at Atomic ResolutionTo elucidate the exact coordination pattern of metal ions is a highly challenging but central goal of our research: Based on the solved NMR structures under AIMs A and B, we apply further methods to understand metal ion binding in detail and to correlate it directly to structural changes and possibly catalysis of the RNAs. In combination with the Metal Ions in Nucleic Acids (MINAS) database, this will allow us to establish general rules for the roles of metal ions in nucleic acids.
-