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Photo-cross-linking mass spectrometry for single amino acid assignment of RNA-binding sites in RNA-binding proteins

ABSTRACT: Mass spectrometry data of peptide–RNA cross-links derived from complex biological systems. Yeast (pre-)mRNPs were isolated by TAP tag purification of Cbp20; human protein–RNA complexes were assembled on an aptamer tagged pre-mRNA. Isolated complexes were UV irradiated. For each of the aforementioned experiments, a non-irradiated control was prepared. Yeast RBPs cross-linked in vivo after labeling with 4-thio-uridine (4SU) were isolated with oligo d(T). All samples were enriched for cross-linked heteroconjugates prior to LC-MS analysis.

INSTRUMENT(S): LTQ Orbitrap Velos

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

SUBMITTER: Timo Sachsenberg

LAB HEAD: Oliver Kohlbacher

PROVIDER: PXD000513 | Pride | 2014-08-07

REPOSITORIES: Pride

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Source:

			Action	DRS
	XL_human_RBPs_UV.IdXML	Other
	XL_human_RBPs_UV.mzML	Mzml
	XL_human_RBPs_control.idXML	Other
	XL_human_RBPs_control.mzML	Mzml
	XL_yeast_RBPs_Ex10_UV.IdXML	Other

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Project description:Ultraviolet (UV) wavebands in sunlight are immunomodulatory. About half the amount of UVA within a minimum erythemal dose of sunlight is systemically immunosuppressive, while higher doses protect from UVB immunosuppression in mice. We have previously shown that these responses to UVA are genetically restricted as they occur in C57BL/6 but not Balb/c mice. We used gene set enrichment analysis of microarray data and real-time RT-PCR confirmation to determine the molecular mechanisms associated with UVA immunomodulation. We found up-regulation of mRNA for the alternative complement pathway. The core-enriched genes complement component 3, properdin and complement factor B were all activated by the immunosuppressive dose of UVA only in UVA-responsive C57BL/6 but not unresponsive BALB/c mice. This therefore matched the genetic restriction and dose responsiveness of UVA immunosuppression. The immune-protective higher UVA dose prevented UVB from down regulating chemokine receptor 7 and IL-12B, and decreased IL-10, supporting previous identification of IL-12 and IL-10 in high dose UVA protection from UVB immunosuppression. Our study has identified activation of the alternative complement pathway as a trigger of UVA-induced systemic immunosuppression and suggests that this pathway is likely to be an important sensor of UVA-induced damage to the skin. 24 hours after UVA, UVB and ssUV irradiation, a 1 cm2 uniform section of skin was excised from the dorsal surface of irradiated and control mice. Total RNA was then extracted from the whole skin using TRIzol reagent (Gibco Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturerâ??s instructions, purified, DNase treated and reverse transcribed into cDNA. For the microarray study a direct incorporation of Cyanine 3-dCTP and Cyanine 5-dCTP fluorescent dyes (Perkin Elmer Life Sciences, Inc. Boston, MA, USA) was used for cDNA synthesis. For each UV dose, a reference design was used to compare an unirradiated control against an irradiated sample. Microarray experiments used compugen 22k mouse oligonucleotide microarray slides (The Clive and Vera Ramaciotti Centre for Gene Function Analysis, Sydney Australia (http://www.ramaciotti.unsw.edu.au). Lower and higher UVA doses were used. C57BL/6 mice were irradiated with lower UVA, higher UVA, UVB, or ssUV; Balb/C mice were irradiated with lower or higher UVA. Experiments were replicated 6 times for each UV dose. A fluorescent dye swap was done for each alternate hybridisation to reduce systematic dye bias of incorporated fluorescent dyes.

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Photo-cross-linking mass spectrometry for single amino acid assignment of RNA-binding sites in RNA-binding proteins

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