PXD024010

PXD024010 is an original dataset announced via ProteomeXchange.

Title	Single Nucleotide Resolution RNA-Protein Cross-Linking/MS: Simple Extension of the CLIR-MS Workflow
Description	RNA-protein interactions mediate a vast number of intracellular processes. CLIR-MS (cross-linking of isotope labeled RNA and tandem mass spectrometry) is a mass spectrometric technique that allows the identification of RNA-protein interaction sites at single nucleotide/amino acid resolution in a single experiment. The use of isotopically labeled RNA segments for UV light induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, thus increasing resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g. in cell or tissue samples. A straightforward approach that maintains the advantages of isotopic labeling but obviates the need for segmental RNA labeling would therefore advance the field. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during the cross-linking reaction and subsequently adds an isotopic label during sample preparation for MS analysis. The approach uses commercially available reagents and can be performed without specialized equipment in any lab. After RNase and protease digests of a cross-linked complex, an RNA-peptide adduct consists of a single peptide and a short nucleic acid adduct. We label the nucleic acid part of these adducts using the enzyme T4 polynucleotide kinase (T4-PNK) and a 1:1 mixture of heavy (18O4-gamma-ATP) and light ATP. In this simple, one-step reaction three of the four heavy oxygen atoms are transferred from the gamma-phosphate to the 5'-end of the RNA adduct. The isotopic difference of light and heavy cross-linked peptides (6.01 Da) can be detected using tandem mass spectrometry after enrichment of the cross-linked peptides. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element RNA (FBE-RNA). Using a variation of the approach, we were able to label a single phosphate within an RNA and unambiguously determine the cross-linking site of the FOX1-RRM to the FBE at single residue resolution on RNA- and protein level. Specifically introducing isotopic labels improves identification of cross-linked species and enables relative quantification based on isotope dilution.
HostingRepository	PRIDE
AnnounceDate	2021-11-04
AnnouncementXML	Submission_2021-11-04_00:32:03.965.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Michael Götze
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	(18)O label at both C-terminal oxygens
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2021-02-05 03:03:52	ID requested
⏵ 1	2021-11-04 00:32:04	announced

Dataset with its publication pending

submitter keyword: Protein RNA cross-linking CLIR-MS UV XL

Alexander Leitner
contact affiliation	Institute of Molecular Systems Biology, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland
contact email	leitner@imsb.biol.ethz.ch
lab head
Michael Götze
contact affiliation	Department of Biology
contact email	mgoetze@ethz.ch
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD024010
     1. Label: PRIDE project
     2. Name: Single Nucleotide Resolution RNA-Protein Cross-Linking/MS: Simple Extension of the CLIR-MS Workflow