Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

Abstract

RNA−protein interactions mediate many intracellular processes. CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA−protein interaction sites at single nucleotide/amino acid resolution in a single experiment. Using isotopically labeled RNA segments for UV-light-induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, increasing spatial 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. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during cross-linking and subsequently adds an isotopic label during sample preparation for MS analysis. After RNase and protease digests of a cross-linked complex, the nucleic acid part of a peptide−RNA conjugate is labeled using the enzyme T4 polynucleotide kinase and a 1:1 mixture of heavy 18 O 4 -γ-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the γphosphate to the 5′-end of the RNA, introducing an isotopic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOXbinding element (FBE) RNA. We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution. Data are available via ProteomeXchange with the identifier PXD024010.

Nucleotide-amino acid π-stacking interactions initiate photo cross-linking in RNA-protein complexes

“…We introduce 13 C-labelled ribonucleotides into the FOX RBE heptanucleotide and use CLIR-MS to identify RNA-protein cross-links with site-specific resolution. Cross-linking on the protein clusters at amino acids around two phenylalanines, consistent with previous findings 37 . However, with few exceptions, it only occurs on the RNA at U 1 , G 2 and G 6 .…”

“…1c ). Sites of cross-linking at the protein were centered at two phenylalanines (F126 and F160), with a distribution of 1-3 amino acids flanking these positions 37 . This was confirmed from analysis of the MS/MS spectra in which fragment ions localize the RNA adducts unambiguously on the peptide backbone (Fig.…”

“…After irradiation of the RNA-protein complex, and controlled digestion to nucleotide-peptide adducts, the locations of cross-linked nucleotides are pinpointed site-specifically. We demonstrated this methodological advance with a study of the interaction of the RRM domain of the RBFOX family bound to its consensus binding element (U)GCAUGU, for which we have previously determined an NMR structure 36 and studied cross-linking 37 . Photo-irradiation of the FOX RRM -FOX RBE complex led to key observations with potentially wide-ranging implications: 1) strong cross-linking occurred between U 1 , G 2 and G 6 with clusters of amino acids centred around the phenylalanines F126 and F160; 2) very little cross-linking was observed at other uridines in the parent or a mutated FOX RBE ; and 3) amino acids that flank F126 and F160 also cross-linked efficiently to U 1 , G 2 and G 6 , but not to other nucleotides of FOX RBE .…”

“…CLIR-MS technology is inherently flexible and we are exploring further improvements to the method 37 . However, the method described here requires the use of chemically synthesized, isotope-labelled RNA and is currently restricted to the study of purified individual RNA-protein complexes.…”

Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS

“…Multiple RNA isotope labelling strategies are available for CLIR-MS 75 , and are employed in experiments presented here. 13 C 15 N labelling results from transcription of RNA in isotopically labelled cell culture medium, as demonstrated previously 34 .…”