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Small molecule quercetin binds MALAT1 triplex and modulates its cellular function - PubMed

  • ️Sat Jan 01 2022

Small molecule quercetin binds MALAT1 triplex and modulates its cellular function

Isha Rakheja et al. Mol Ther Nucleic Acids. 2022.

Abstract

The triple-helix structure at the 3' end of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA, has been considered to be a target for modulating the oncogenic functions of MALAT1. This study examines the binding of quercetin-a known triplex binding molecule-to the MALAT1 triplex. By employing UV-visible spectroscopy, circular dichroism spectroscopy, and isothermal titration calorimetry, we observed that quercetin binds to the MALAT1 triplex with a stoichiometry of 1:1 and K d of 495 ± 61 nM, along with a negative change in free energy, indicating a spontaneous interaction. Employing real-time PCR measurements, we observed around 50% downregulation of MALAT1 transcript levels in MCF7 cells, and fluorescence in situ hybridization (FISH) experiments showed concomitantly reduced levels of MALAT1 in nuclear speckles. This interaction is likely a result of a direct interaction between the molecule and the RNA, as indicated by a transcription-stop experiment. Further, transcriptome-wide analysis of alternative splicing changes induced by quercetin revealed modulation of MALAT1 downstream genes. Collectively, our study shows that quercetin strongly binds to the MALAT1 triplex and modulates its functions. It can thus be used as a scaffold for further development of therapeutics or as a chemical tool to understand MALAT1 functions.

Keywords: MALAT1; MT: Oligonucleotides: Therapies and Applications; RNA FISH; in silico docking; isothermal titration calorimetry; quercetin; small-molecule binding; triple helix.

© 2022 The Author(s).

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1

MALAT1 triplex sequence and control sequence along with the chemical structure of quercetin (A) Structure of the last 94 bp at the 3′ end of the MALAT1 RNA (complete triplex region). Nucleotide base numbers are marked. (B) The 73-nt control sequence with missing second strand, which does not form the triple helix. (C) The chemical structure of quercetin.

Figure 2
Figure 2

CD signatures between MALAT1 triplex and control non-triplex-containing sequences are different (A) CD spectra of MALAT1 triplex (blue) and control duplex (red), both at 2 μM, shows a pronounced triplex-forming signature in the case of the 94-nt triplex. (B) Thermal melting profiles of MALAT1 triplex (blue) and control duplex (red) show characteristic melting temperatures of the MALAT1 triplex. (C) First derivative curves of the raw absorbance signal of MALAT1 triplex (blue) and control duplex (red) along with fitted curves depict different melting temperatures of the respective structural components. Experiments were performed three times in 10 mM sodium cacodylate buffer (pH 7) in the presence of 100 mM NaCl and 0.5 mM MgCl2. The plotted curves are representative of the same.

Figure 3
Figure 3

UV titration of quercetin with triplex and control sequences Absorbance spectra of 0.5 μM quercetin with successive additions of (A) MALAT1 triplex and (B) control duplex in 10 mM sodium cacodylate (pH 7), 0.5 mM MgCl2, and 150 mM NaCl at 25°C. (C) Variation in absorbance at 372 nm as a function of quercetin concentration for the triplex (□) and the control (○) structures shows a sharper rate of decline in absorbance for the triplex-forming sequence compared with the 73-nt control sequence, indicating a stronger interaction of quercetin with the 94-nt triplex sequence.

Figure 4
Figure 4

Binding parameters of quercetin to the MALAT1 triplex indicate clear binding to the MALAT1 triplex sequence Thermograms for the calorimetric titration of 100 μM quercetin in 10 mM sodium cacodylate (pH 7), 0.5 mM MgCl2, and 150 mM NaCl into (A) buffer or (B) 6 μM 94-nt complete triplex or (C) 73-nt control structure at 25°C indicate binding of quercetin with the triplex. (D) Plot of integrated heats versus quercetin to triplex (□) molar ratio and duplex (○) molar ratio indicates no binding to quercetin in the case of control duplex and clear binding in the case of triplex. The first data point was eliminated in the data fit.

Figure 5
Figure 5

In silico docking of quercetin against the MALAT1 triplex (A) Docking of quercetin to the MALAT1 triple-helix crystal structure. The flanking strands are colored in sea green (49–59) and golden yellow (7–16), while the middle strand is colored in purple (83–93). The interacting nucleotides are colored in pink. (B) The lowest-energy docking pose of the ligand (represented in red). (C) Atomistic representation of the ligand bound to the MALAT1 triplex. The ligand is depicted in yellow bonds, while the bonds of the lncRNA are shown in brown. Carbon, oxygen, nitrogen, and phosphorus atoms are depicted in black, red, blue, and purple, respectively. The hydrogen bonds between the ligand and the receptor are shown as a green dashed line with the distance marked in angstroms. The nucleotides involved in the hydrophobic interaction are shown with a red sun-flare semicircle. (The nucleotide residue numbers in all images correspond to the numbering of the 94-nt MALAT1 triplex as shown in Figure 1.)

Figure 6
Figure 6

Change in transcript levels upon quercetin treatment Quantitative real-time PCR showing (A) MALAT1 expression levels in MCF7 cells upon treatment with the indicated concentrations of the small molecule quercetin for 24 h. (B) Fold change in NEAT1 levels relative to control upon 1 μM quercetin treatment for 24 h in MCF7 cells. (C) Transcription-stop assay using α-amanitin. Bars depict fold change in levels of MALAT1 lncRNA after treatment with quercetin. The x axis shows time following quercetin treatment. All results presented are an average of three biological replicates (n = 3). Error bars represent SEM; asterisks denote Student’s two-tailed t test p values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). n.s. refers to non-significant.

Figure 7
Figure 7

MALAT1 RNA puncta inside cells are reduced upon quercetin treatment Representative images showing (A) untreated vs. (B) 1 μM quercetin-treated cells. The left shows MALAT1 puncta (green) alone and the right shows MALAT1 puncta along with DAPI (blue). Scale bars, 10 μm. (C) Quantification of MALAT1 puncta (of size 1–1,000 pixels) per cell per field, counted for at least 100 cells per treatment; Error bar represents SEM from n=10 replicate fields; asterisk denotes Student’s two-tailed t test p value (∗p < 0.05). (D) Representative images showing the colocalization of MALAT1 and SC35 in MCF7 cells. DNA is stained with DAPI and represented in blue. Scale bar, 5 μm.

Figure 8
Figure 8

Differentially expressed transcripts and distribution of splicing events between DMSO-treated (control) and quercetin-treated MCF7 cells (A) Volcano plot to show differentially expressed transcripts. Dashed lines represent –log10 P and log2 FC cutoff of ±1. (B) Pie chart to show differentially spliced events. Events are denoted as A3, alternative 3′ usage; A5, alternative 5′ end of transcript; AF, alternative first exon; AL, alternative last exon; MX, mutually exclusive usage; RI, intron retention event; SE, exon skipping event.

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