G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme

Abstract

Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms.

Keywords: G-quadruplex; LSD1; RNA–protein interactions; TERRA; binding; chromatin; enzyme; kinetics; lncRNA; mass spectrometry; ncRNA; structure.

FIGURE 1.

TERRA RNA recruits LSD1 to deprotected telomeres. (A) A functional role for the TERRA RNA in the processing of uncapped telomeres, as previously reported (Porro et al. 2014b). TERRA can serve as a scaffold for LSD1–MRE11 associations. (B) Protein constructs used in this study include LSD1 (aa 171–852) and CoREST (aa 286–482 plus 6xHis-tag sequence). LSD1 consists of a SWIRM domain (red), an intertwined monoamine oxidase domain (cyan/blue), and a tower domain (green), based on PDB 2IW5 (Yang et al. 2006). LSD1 biological function requires the presence of CoREST (shown in orange). (C) A GQ RNA is stabilized by specific monovalent ions (including K⁺ and Na⁺ denoted as blue spheres). A single GQ RNA unit and a stacked GQ RNA were prepared to investigate binding affinity and specificity of the GQ RNA–LSD1 interaction. The UUAGGG repeat elements of TERRA can form a stable parallel-stranded GQ RNA in vivo (Xu et al. 2010) and a model of the higher order TERRA RNA architecture has been previously demonstrated (Martadinata and Phan 2013).

FIGURE 2.

Monovalent ions dramatically influence the structure of GQ-forming RNAs. (A) Parallel-stranded GQ RNAs are known to have an Θ_max of ∼263 nm (Balaratnam and Basu 2015). Circular dichroism spectroscopy demonstrates that parallel-stranded GQ structures form in the presence of potassium (black, solid triangle, and circles) and sodium (gray diamond and box), consistent with previous studies. In contrast, lithium (outlined triangles) destabilizes GQ formation. (B) The analysis of analytical ultracentrifugation (AUC) data of (UUAGGG)₄U and (UUAGGG)₈U RNAs in the presence of potassium (K⁺), sodium (Na⁺), and lithium (Li⁺). Figure symbols as in A. The plot shows an overlay of the continuous distribution [C(s)] versus the sedimentation distribution coefficient (S).

FIGURE 3.

Affinity and specificity of LSD1–CoREST binding to distinct nucleic acid structures is dependent on monovalent ions. (A) Analysis of gel-mobility shift assay binding curves of (UUAGGG)₈U, (UUAGGG)₄U, and 25-nt ssRNA. Assays were performed in potassium (K⁺), sodium (Na⁺), and lithium (Li⁺) (symbols same as in Fig. 2) using LSD1–CoREST (amino acid residues 171–852 and 286–482, respectively) with exogenous protein purification tags removed. LSD1 strongly prefers to bind stacked GQ-forming RNA structures. The plot shows the fraction of RNA bound at various LSD1–CoREST concentrations (log scale). Error bars for each data point represent the range of three independent experiments. The dissociation constant (K_d) and Hill coefficient (h) from this analysis are reported in Table 1. (B) Representative gels showing that RNA binding activity of LSD1–CoREST is dependent upon the ability to form a GQ RNA conformation. Complexes and free oligonucleotides were resolved on a 0.6% native agarose gel. The concentration of the LSD1–CoREST complex is noted for each lane (nM).

FIGURE 4.

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.

FIGURE 5.

Location of an ssRNA binding region on LSD1. (A) Analysis of crystal soaks of a 5′-UUAGG-3′ RNA ligand into LSD1–CoREST crystals shows clear difference electron density (|F_o|−|F_c|) (dark gray mesh contoured at 5.5 r.m.s.d). RNA–LSD1 interactions occur along β-sheet and loop regions of LSD1 (yellow box). LSD1 and CoREST as colored in Figure 1B. (B) The 2.8 Å resolution map reveals the unambiguous identity and directionality of the RNA (pink), shown in stick representation. The difference electron density (|F_o|−|F_c|) is noted (dark gray mesh contoured at 5.5 r.m.s.d) and nucleobase density was observed for UUAG nucleotides. (C) Schematic of noncovalent interactions between the ssRNA fragment and LSD1. Dotted lines indicate RNA–LSD1 residues within 3.0 Å (as summarized in Supplemental Table 2), with conserved LSD1 residues (bold) spanning the monoamine oxidase domain (cyan/blue). (D) A close-up view of the difference density (|F_o|−|F_c|) shows the unambiguous assignment of purine (A, G) nucleobases.

FIGURE 6.

Model of the TERRA RNA–LSD1 binding interface. Cross-linking mass spectrometry data support a model whereby LSD1 residues (red) in the SWIRM domain interact with a stacked GQ RNA. In addition, X-ray crystallography data identifies an ssRNA binding location (pink), which resides adjacent to the GQ RNA binding site. Binding locations are consistent with a GQ RNA that binds LSD1 in a noncompetitive manner.