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  Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions.

Zaman, U., Richter, F. M., Hofele, R. V., Kramer, K., Sachsenberg, T., Kohlbacher, O., et al. (2015). Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions. Molecular and Cellular Proteomics, 14(12), 3196-3210. doi:10.1074/mcp.M115.052795.

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 Urheber:
Zaman, U.1, Autor           
Richter, F. M., Autor
Hofele, R. V.1, Autor           
Kramer, K.1, Autor           
Sachsenberg, T., Autor
Kohlbacher, O., Autor
Lenz, C.1, Autor           
Urlaub, H.1, Autor           
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              

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 Zusammenfassung: Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C4H8S2O2) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.

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Sprache(n): eng - English
 Datum: 2015-10-082015-12-01
 Publikationsstatus: Erschienen
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 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: DOI: 10.1074/mcp.M115.052795
 Art des Abschluß: -

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Titel: Molecular and Cellular Proteomics
Genre der Quelle: Zeitschrift
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Seiten: - Band / Heft: 14 (12) Artikelnummer: - Start- / Endseite: 3196 - 3210 Identifikator: -