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Bottom-up high-resolution RNA mass spectrometry

English title Bottom-up high-resolution RNA mass spectrometry
Applicant Leidel Sebastian
Number 198515
Funding scheme R'EQUIP
Research institution Departement für Chemie, Biochemie und Pharmazie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biochemistry
Start/End 01.10.2021 - 30.09.2022
Approved amount 525'000.00
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All Disciplines (2)

Discipline
Biochemistry
Molecular Biology

Keywords (5)

RNA; mass spectrometry; nucleic acids; systems biology; chemical modification

Lay Summary (German)

Lead
Zelluläre RNA Moleküle spielen eine entscheidende Rolle in allen Prozessen des Lebens; sowohl in gesunden Zellen als auch solchen, deren Funktionalität gestört ist. Interessanterweise werden viele der RNA Moleküle nach deren Herstellung chemisch modifiziert. Dafür bedienen sich die Zellen einer Vielzahl von Modifizierungsenzymen, die in allen Zelltypen und Organismen vorkommen. In den letzten Jahren hat sich zunehmend gezeigt, dass besonders in einer Reihe neurodegenerativen Erkrankungen und Krebs die Modifikationsmuster von RNA stark vom Normalzustand abweichen können.
Lay summary

Inhalt und Ziel des Forschungsprojekts

Das übergeordnete Ziel dieses Projektes ist es, Methoden zu etablieren, um die hochauflösende und empfindliche Charakterisierung von RNA-Modifizierungen zu ermöglichen. Dazu wird Massenspektrometrie als Methode angewandt, da diese Strategie momentan die einzige Möglichkeit ist, Modifizierungen zweifelsfrei und direkt nachzuweisen.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Unsere Arbeit wird neue Nachweismethoden für RNA Modifizierungen entwickeln und dadurch Erkenntnisse zum Vorkommen und der Rolle dieser Modifizierungen in unterschiedlichen Kontexten gewinnen. Diese Erkenntnisse dienen sowohl dem Ziel der Grundlagenforschung als auch der Untersuchung komplexer degenerativer Erkrankungen und Krebs.

Direct link to Lay Summary Last update: 13.11.2020

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
184947 The role of tRNA modifications in vertebrates 01.01.2020 Project funding (Div. I-III)
196387 A multidisciplinary approach to identify vulnerabilities of SARS-CoV-2 for vaccine development 01.06.2020 Special Call on Coronaviruses

Abstract

Cellular RNA molecules contain a plethora of chemical nucleoside modifications. This phenomenon has recently gained a lot of attention, because first, these posttranscriptionally added modifications extend the chemical space of RNA thereby enabling new functionalities. Second, RNA modifications can tune the activity, stability and turnover of RNA molecules. Third, a subset of RNA modifications is reversible and respond to diverse stimuli. This constitutes a new layer of posttranscriptional regulation, termed “epitranscriptomics”. Finally, genetic studies have identified numerous RNA modification defects that are linked to human diseases in particular neuronal degeneration and cancer. And even though nearly every cellular RNA molecule is modified, the mechanistic role of chemical RNA modifications during development and disease is still poorly understood. Therefore, methods that enable their sensitive and accurate identification and quantification are crucial to close this gap of knowledge.While sequencing-based strategies have made a lot of progress, these methods are rarely quantitative and do not provide direct evidence for the presence of a modification. Therefore, RNA mass spectrometry is the gold standard to directly identify and quantify nucleoside modifications as it allows for the unequivocal identification of specific modifications and their sensitive quantification.My laboratory uses RNA mass spectrometry since 2010 to develop strategies for the sensitive analysis of biological samples using an orbitrap-based instrument. By developing such tools, my vision is to remove the limitations that prevent potential users from applying RNA mass spectrometry. Therefore, we use instruments that are found in most proteomics facilities and we develop software to enable users to analyze their data independently. In 2019 I moved to the Department of Chemistry and Biochemistry (DCB) of the University of Bern, where I was recruited to a new professorship for RNA biochemistry in the context of the NCCR RNA & Disease. To establish and to further develop our workflow for RNA mass spectrometry at the DCB, I would like to acquire a new Orbitrap Exploris 480. This highly versatile instrument will allow us to first, push the sensitivity and accuracy of our analysis to the next level, second we will use it to establish new sequence-specific assays and finally, we will use it to perform top-down analyses of intact RNA molecules like tRNA and miRNA. This will give us unprecedented insights into the modification spectrum of cellular RNA. Importantly, 23 research projects will directly benefit from the instruments. 19 of these projects are conducted outside the DCB. The project partners include 17 laboratories outside the University of Bern in all regions and major academic centers of Switzerland. The instrument will therefore serve a strong need of users that is currently unmet. The project is strongly supported by the NCCR RNA and Disease, by the DCB and by the University of Bern. Finally, several excellent researchers, who did not have concrete projects but emphasize that the availability of the technology is crucial for Swiss RNA research have written additional support letters.
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