G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme
- Alexander Hirschi1,3,
- William J. Martin1,3,
- Zigmund Luka1,
- Lioudmila V. Loukachevitch2 and
- Nicholas J. Reiter1
- 1Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0146, USA
- 2Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA
- Corresponding author: nick.reiter{at}vanderbilt.edu
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↵3These authors contributed equally to this work.
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
- LSD1
- TERRA
- lncRNA
- ncRNA
- G-quadruplex
- chromatin
- enzyme
- kinetics
- binding
- mass spectrometry
- structure
- RNA–protein interactions
Footnotes
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Abbreviations: LSD1, lysine-specific histone demethylase 1; CoREST, co-repressor for repressor element 1 silencing transcription factor; diMeK4H31-21, dimethyl-Lys4 Histone H3 peptide aa1-21; 4-AAP, 4-aminoantipyrine; DCHBS, 3,5-dichloro-2-hydroxybenzenesulfonic acid; HRP, horseradish peroxidase; EDTA, ethylenediaminetetraacetic acid; TB, Tris–Borate; GST, glutathione S-transferase; GQ, G-quadruplex; TERRA, telomeric repeat containing RNA; HOTAIR, homeotic transcript antisense RNA; p53, tumor suppressor p53; E2F1, E2F transcription factor 1; DNMT1, DNA cytosine-5-methyltransferase 1; MRE11, meiotic recombination 11; HDAC1/2, histone deacetylase 1/2; nt, nucleotide; lncRNA, long noncoding ribonucleic acid
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.057265.116.
- Received May 2, 2016.
- Accepted May 5, 2016.
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