Single Nucleotide Resolution RNA-Protein Cross-Linking/MS: Simple Extension of the CLIR-MS Workflow

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michael Götze  

LAB HEAD: Alexander Leitner

PROVIDER: PXD024010 | Pride | 2021-11-04

REPOSITORIES: Pride