A highly sensitive protein-RNA cross-linking mass spectrometry workflow with enhanced structural modeling potential
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2025-06-24
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Journal Article
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yes
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Abstract
Protein-RNA interactions underpin many critical biological processes, demanding the development of technologies to precisely characterize their nature and functions. Many such technologies depend upon cross-linking under mild irradiation conditions to stabilize contacts between amino acids and nucleobases; for example, the cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) method. A deeper understanding of the CLIR-MS workflow is required to maximize its impact for structural biology, particularly addressing the low abundance of cross-linking products and the information content of spatial/geometric restraints reflected by a cross-link. Here, we present a vastly improved CLIR-MS pipeline that features enhanced sample preparation, data acquisition and interpretation. These advances significantly increase the number of detected cross-link products per sample. We demonstrate that the procedure is robust against variation of key experimental parameters, including irradiation energy and temperature. Using this improved protocol on four protein-RNA complexes representing canonical and non-canonical RNA-binding domains, we propose for the first time the distances encoded by protein-RNA cross-links, enabling their use as structural restraints. We also compared the cross-linking of canonical RNA with 4-thiouracil-labeled counterparts, showing slight, but noticeable differences. The improved understanding of protein-RNA cross-links refines their structural interpretation and facilitates the adoption of the method in integrative/hybrid structural biology.
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53 (11)
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Oxford University Press
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03591 - Allain, Frédéric / Allain, Frédéric
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ETH-24 16-2 - A new technology for the structural analysis of protein-RNA interactions at single residue resolution and its application to understand pre-miRNA processing in vitro and in vivo. (ETHZ)
670821 - Proteomics 4D: The proteome in context (EC)
182880 - NCCR RNA#38;Disease (51NF40-182880): Flexibility Grant (SNF)
115766 - Unrestricted Leveraging of Targets for Research Advancement and Drug Discovery (EC)
670821 - Proteomics 4D: The proteome in context (EC)
182880 - NCCR RNA#38;Disease (51NF40-182880): Flexibility Grant (SNF)
115766 - Unrestricted Leveraging of Targets for Research Advancement and Drug Discovery (EC)