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

ABSTRACT: In the present paper, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. To prove this three model systems, a) a small synthetic peptide from smB'/B protein incubated with U1snRNA oligonucleotide, b) in vitro reconstituted 15.5K protein with U4 snRNA oligonucleotide and c) native ribonucleoprotein-complexes (RNPs) from S. cerevisiae were used . All protein-RNA complexes were UV irradiated and heteroconjugates cross-linked enriched prior to LC-MS analysis. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks which is observable as a mass increments of 152 Da upon mass spectrometric analysis.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Uzma Zaman

LAB HEAD: Henning Urlaub

PROVIDER: PXD002656 | Pride | 2015-10-13

REPOSITORIES: pride

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			Action	DRS
	XL_U4_Ex1_C13ATP.csv	Csv
	XL_U4_Ex1_C13ATP.idXML	Other
	XL_U4_Ex1_C13ATP.mzML	Mzml
	XL_U4_Ex1_C13ATP.raw	Raw
	XL_U4_Ex1_C13ATP.xlsx	Xlsx

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Publications

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein-RNA Interactions.

Zaman Uzma U Richter Florian M FM Hofele Romina R Kramer Katharina K Sachsenberg Timo T Kohlbacher Oliver O Lenz Christof C Urlaub Henning H

Molecular & cellular proteomics : MCP 20151008 12

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 ...[more]

PMID: 26450613

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

Project description:In the present paper, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. To prove this three model systems, a) a small synthetic peptide from smB'/B protein incubated with U1snRNA oligonucleotide, b) in vitro reconstituted 15.5K protein with U4 snRNA oligonucleotide and c) native ribonucleoprotein-complexes (RNPs) from S. cerevisiae were used . All protein-RNA complexes were UV irradiated and heteroconjugates cross-linked enriched prior to LC-MS analysis. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks which is observable as a mass increments of 152 Da upon mass spectrometric analysis.

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