G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.
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.
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0146, USA.
Alexander
A
Hirschi
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0146, USA.
William J
WJ
Martin
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0146, USA.
Zigmund
Z
Luka
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Lioudmila V
LV
Loukachevitch
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0146, USA.
Nicholas J
NJ
Reiter
D054856:G-Quadruplexes; D056466:Histone Demethylases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*
D012313:RNA; D056466:Histone Demethylases; D008239:Lysine
G-quadruplex; LSD1; RNA–protein interactions; TERRA; binding; chromatin; enzyme; kinetics; lncRNA; mass spectrometry; ncRNA; structure