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Principles of Metal-Ion-Assisted Folding and Structure of Functional RNAs

English title Principles of Metal-Ion-Assisted Folding and Structure of Functional RNAs
Applicant Sigel Roland K. O.
Number 192153
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 01.04.2020 - 31.03.2024
Approved amount 1'004'879.00
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All Disciplines (2)

Discipline
Inorganic Chemistry
Biophysics

Keywords (7)

Coordination Chemistry; Regulatory RNA; Riboswitches; Metal Ions; Ribozymes; smFRET; NMR

Lay Summary (German)

Lead
Ribonucleinsäuren (RNA) sind essentiell für jedes Lebewesen. Regulatorische RNAs kontrollieren Proteinexpression, deren Transport, oder auch den Lebenszyklus einer Zelle, indem sie die Transkription und/oder Translation entsprechender Gensequenzen regulieren. Andere RNAs sind katalytisch aktiv (Ribozyme), ähnlich wie Enzyme. Faltung, Struktur, und Aktivität dieser funktionellen RNAs hängt dabei von deren Nukleotidsequenz, aber vorallem auch von verschiedenen zusätzlichen Faktoren, hauptsächlich Metallionen und assoziierte Proteine, ab. Im Falle von regulatorischen Riboschaltern (riboswitch) binden z.B. metallbasierte Metabolite wie der Molybdenkofaktor (Moco) spezifisch und führen zu einer Änderung der 3D-Struktur als Mittel der Genregulation.
Lay summary

Ziel dieses Projektes ist es, die metallionenkontrollierte Struktur-Funktion-Beziehung von drei wichtigen RNA-Klassen aufzuschlüsseln und zu verstehen: (i) dem bakteriellen Moco Riboschalter, (ii) dem CPEB3, das einzige bekannte Ribozym in Säugetieren, und verwandten katalytischen RNAs, sowie (iii) einem selbst-spleissenden Gruppe II Intron, welches als Vorläufer des grössten Teils des menschlichen Genoms gilt. Ziel von (i) ist es die Struktur und Mechanismus dieses einzigartigen Riboschalters und somit auch die Regulation der in die Moco Biosynthese involvierten Enzyme zu verstehen. Die kleinen "Hepatitis-Delta-ähnlichen" Ribozyme in (ii) bieten ein hervorragendes System zur Untersuchung der komplizierten Wechselwirkung zwischen Metallionen, Faltung, Dynamic, Struktur und Mechanismus von katalytischen RNAs.  Im dritten Teil (iii) liegt der Fokus auf dem Verständnis der Kofaktor-Rolle von möglichen RNA-bindenden Proteinen in vitro und in der Zelle.

Durch eine einzigartige Kombination biochemischer und molekularbiologischer Methoden mit klassischer Koordinationschemie, sowie biomolekularer NMR- und Einzelmolekülspektroskopie können wir solche komplexen RNA Systeme detailliert in Bezug auf Struktur, Bindungs- und Faltungsgleichgewichte, sowie Dynamik untersuchen und verstehen. Angesiedelt in der klassischen Bioanorganischen Chemie, hat dieses Projekt auch einen grossen Einfluss auf die RNA Biochemie, Strukturbiologie, und Medizinische Chemie.

Direct link to Lay Summary Last update: 28.03.2020

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Associated projects

Number Title Start Funding scheme
124834 Coordination Chemistry within the Core of Large RNAs: Regulating Tertiary Contacts and Function 01.04.2009 Project funding (Div. I-III)
143750 Metal Ion-Guided Assembly and Structures of the Catalytic Core of Ribozymes 01.10.2012 Project funding (Div. I-III)
165868 Metal Ions in Structure and Function of Regulatory RNAs 01.04.2016 Project funding (Div. I-III)

Abstract

Only a minority of cellular RNAs are "information-transfer-agents" like mRNA or tRNA. The vast majority are functionally active, e.g. ribozymes perform catalytic reactions and riboswitches regulate the concentration of certain metabolites on the transcriptional and/or translational level. Structure and function of these RNAs is tightly and inescapably linked to metal ions (Mn+) and in many cases function as an RNA-protein complex in vivo. The goal of this project is to understand the folding and structure function relationship with Mn+ and their partial replacement with proteins under more in vivo-like conditions of three key RNAs: (i) the bacterial Moco riboswitch, (ii) HDV-like ribozymes, and (iii) a self-splicing group II intron. AIM 1: Moco Riboswitch: A Redox-Active Metabolite in the Center of an RNA-Protein ComplexThe molybdenum redox cofactor Moco-sensing riboswitch is still to a large part a myth. This riboswitch regulates enzymes of Moco biosynthesis. We have established the necessary procedures to perform all experiments under the exclusion of O2 and now aim to understand in detail this riboswitch. The O2-sensitivity of the Moco makes this project highly challenging.AIM 2: Structure and Mechanism of Mammalian and Microbial HDV-like RibozymesThe CPEB3 ribozyme is the only confirmed ribozyme in mammals aside from the ribosome and the spliceosome. This hepatitis delta virus (HDV)-like RNA is located in an intronic sequence within the cytoplasmatic polyadenylation element-binding protein 3. Its single-nucleotide-polymorphism is linked to cleavage activity and the performance of our episodic memory. Here we aim to investigate the structure-function relationship of HDV-like ribozymes by X-ray, NMR, and further methods to understand the interplay between Mn+, folding, dynamics, structure and mechanism. AIM 3: Replacing Mn+ by Proteins in In Vivo Group II Intron FoldingGroup II introns and the nuclear spliceosome are evolutionary related. Recent studies propose that the spliceosome has gradually evolved from group II introns, but so far, there is no experimental evidence for this hypothesis. Generally, Structural Biology provides us with an enormous knowledge about the architecture of biological scaffolds in vitro, but corresponding data within the natural in vivo environment are largely lacking. Here we follow a systematic approach towards the in vivo studies of this system by taking an engineered RNA construct and bringing it back to its origin. Applying fluorescence spectroscopy either in the ensemble or on a single-molecule level, we will provide first evidence of how group II introns have been developed into the spliceosome, invaded the nuclei, and elucidate the way by which protein cofactors replace the function of metal ions in vivo.To reach the above three goals, we employ a combination of biochemical tools, coordination chemistry, biomolecular NMR spectroscopy, Xray crystallography and single molecule (sm)FRET. This interdisciplinary approach that we have been following for years, successfully provides us with a unique and exceptional possibility to understand such complex RNAs in detail with respect to structure, thermodynamics and kinetics. Although this project is centered in the classical field of Biological Inorganic Chemistry, it will also have a great impact on related fields like RNA Biochemistry, Structural Biology, and Medicinal Chemistry.
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