Identification of the G-quadruplex RNA binding domain of LSD1. The location of the LSD1 region that cross-links with the GQ RNA was detected via mass spectrometry. (A) Relative coverage of LSD1 residues upon GQ RNA cross-linking. UV light was used to cross-link biotinylated GQ RNA with LSD1–CoREST and the covalent complex was purified using streptavidin beads. LSD1 was then analyzed with mass spectroscopy alongside a control sample that had been treated with UV light in the absence of RNA. A plot of the signal intensity ratio between the control and GQ RNA cross-linked sample reveals peptide fragments that are strongly depleted in the cross-linked sample, likely due to the change in m/z ratio upon formation of RNA adduct. (B) Analysis of the elution profile reveals a depletion of the peptide signal upon cross-linking for a region in the SWIRM domain (227–251). This peptide is dramatically depleted (∼10,000-fold weaker) compared with the control sample. The isotopic distribution (M, M + 1, M + 2, M + 3) confirms the peptide identity, with an isotope dot product (idotp) of 1.00 and 0.97 for the control and cross-linked samples, respectively. (C) The locations of the GQ RNA binding regions are mapped onto the structure of LSD1–CoREST (PDB 4XBF). Two distinct regions of LSD1 appear to cross-link with GQ RNA. The primary GQ RNA cross-link is located within the SWIRM domain (red) (residues 227–251, 210–216) and a minor RNA–LSD1 adduct region exists adjacent to the active site and FAD (brown spheres) and close to the C-terminal domain of CoREST (green) (residues 527–550). Additional GQ RNA–LSD1 cross-link locations are noted (Results and Supplemental Material) but were not consistent between the two separate and independent cross-link-MS experiments.
