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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: PXD002392 | Pride | 2022-03-01

REPOSITORIES: Pride

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			Action	DRS
	XL_U4_EX_1.mzML	Mzml
	XL_U4_EX_C.mzML	Mzml
	XL_U4_EX_G.mzML	Mzml
	XL_U4_Ex_1.idXML	Other
	XL_U4_Ex_A.idXML	Other

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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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