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Revealing the organization and regulation of protein-RNA complexes with structural proteomics methods

English title Revealing the organization and regulation of protein-RNA complexes with structural proteomics methods
Applicant Leitner Alexander
Number 200679
Funding scheme Project funding (Div. I-III)
Research institution Institut für Molekulare Systembiologie ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Biochemistry
Start/End 01.06.2021 - 31.05.2025
Approved amount 560'928.00
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Keywords (9)

protein-RNA interactions; protein complexes; protein-protein interactions; structural biology; cross-linking; mass spectrometry; structural proteomics; systems biology; proteomics

Lay Summary (German)

Lead
Biologische Systeme sind modular aufgebaut und bestehen aus Netzwerken von verschiedenen Klassen von Biomolekülen, wie zum Beispiel Proteinen und Ribonukleinsäuren (DNA und RNA). Diese Biomoleküle liegen vielfach in Komplexen vor, die wichtige zelluläre Prozesse kontrollieren. Protein-RNA-Komplexe sind noch nicht so gut erforscht wie Komplexe, die nur aus Proteinen bestehen. In diesem Projekt wollen wir die Organisation und Regulation von solchen Komplexen in humanen Zellen mit Hilfe von massenspektrometrischen (MS) Methoden untersuchen.
Lay summary

Das Projekt kombiniert drei verschiedene analytische Methoden, um Protein-RNA-Komplexe besser strukturell zu charakterisieren: Die Trennung solcher Komplex mittels Grössenausschlusschromatographie, die Strukturanalyse von Proteinkomponenten mit Hilfe von chemischer Quervernetzung (Cross-linking), und die Vernetzung von Proteinen und RNA durch ultraviolettes Licht. Diese Methoden dienen dazu, um herauszufinden, welche Komplexe sowohl Proteine als auch RNA enthalten, und wie die Bildung solcher Komplexe und deren Aufbau durch zellulären Stress (zum Beispiel ausgelöst durch hohe Temperaturen oder chemische Wirkstoffe) beeinflusst wird.

Das Projekt wird wichtige neue Erkenntnisse darüber liefern, wie zelluläre Netzwerke, die durch die Wechselwirkungen von zwei verschiedenen Klassen von Biomolekülen kontrolliert werden, aufgebaut sind und sich in verschiedenen biologischen Zuständen in ihrer Organisation verändern. Diese Informationen helfen einerseits, grundlegende biologische Prozesse besser zu verstehen, andererseits können sie mögliche neue Ansätze liefern, um diese Prozesse zu steuern.

Direct link to Lay Summary Last update: 13.07.2021

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
204920 Hybrid structural investigation of protein-RNA interactions in the context of RNA-induced liquid-liquid phase separation 01.01.2022 Project funding (Div. I-III)
182880 NCCR RNA & disease: The role of RNA biology in disease mechanisms (phase II) 01.05.2018 National Centres of Competence in Research (NCCRs)
198253 Rapid Hybrid Structure Determination of Coronavirus Protein-RNA Complexes as a Basis for Drug Screening for the Treatment of COVID-19 01.10.2020 NRP 78 Covid-19

Abstract

Biological systems are organized in a modular fashion, consisting of intricate networks of different classes of biomolecules that interact with each other not only as individual molecules, but most often function in higher-level structures such as complexes. Formation of such complexes is particularly important for proteins that may associate with other proteins, but also with other types of biomolecules such as RNA to control elementary functions of the cell. Mass spectrometry-based proteomics has been a crucial technology to study the protein composition of cellular systems, their quantitative changes, and their interactions. More recently, structural mass spectrometry methods have evolved to probe protein complexes, however, despite the important role of protein-RNA interactions, applications to ribonucleoproteins have been relatively limited.In this project, we will elucidate the organization and regulation of protein-protein and particularly protein-RNA complexes in human cell lines by applying structural proteomics techniques at large scale. For the first time, we will combine three mass spectrometry-based methods to study the composition and structure of protein-protein and protein-RNA complexes: (1) Size exclusion chromatography fractionation of complexes under native conditions in combination with data-independent acquisition mass spectrometry, (2) chemical protein-protein cross-linking, and (3) protein-RNA cross-linking induced by ultraviolet light. Both cross-linking strategies will be initially coupled to data-dependent acquisition mass spectrometry. We will apply these methods to two different types of system-wide perturbations: (1) RNase treatment at the cell lysate level will highlight RNA-associated protein complexes in size exclusion chromatography and distinguish them from complexes that do not contain RNA such as pure protein complexes. (2) We will induce cell stress by treatments including heat shock, arsenite or drugs such as thapsigargin at the intact cell level and follow the response of protein-protein and protein-RNA complexes to these perturbations by cross-linking. By combining three structural proteomics methods with systematic perturbation experiments, we will obtain a wealth of proteome-scale data on protein complex organization and regulation, including complex composition, spatial organization of complexes, location of RNA-binding sites, and changes of cellular interaction networks upon perturbation. We will combine these multi-level data to obtain a better understanding of how cells coordinate interactions between proteins and RNA in general and how they organize their response to stress by regulating protein-protein and protein-RNA interactions and the assembly into joint complexes, respectively. Therefore, our structural proteomics approach will generate entirely new insights that are relevant for systems biology, structural biology, and RNA biology.
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