Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA

Abstract

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA-protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.

Figure 1.

Biophysical characterization of MALAT1 rG4 structures. (A–F) CD spectrum under K⁺ and Li⁺ conditions. The CD pattern becomes stronger in the presence of K⁺ rather than Li⁺, suggesting rG4 formation. The negative peak at 240 nm and the positive peak at 262 nm under K⁺ condition suggest a parallel topology of rG4. (G–L) UV melting. Hypochromic shift is observed at 295 nm, a strong sign of rG4 formation. The melting temperatures (Tms) for the MALAT1 rG4s are indicated. (M–R) ThT enhanced fluorescence spectroscopy under K⁺ and Li⁺ conditions with an emission maximum wavelength of 490 nm. The fluorescence intensity of ThT is higher in rG4 under K⁺ compared to Li⁺, verifying the formation of rG4.

Figure 2.

rG4 conservation analysis and predicted G4 scores for MALAT1 rG4s. Comparative sequence analysis of MALAT1 rG4_04 and MALAT1 rG4_09 in different species and their predicted G4 scores. All G4 scores are higher than the threshold set in corresponding G4 prediction programs, i.e. cGcC > 4.5, G4H > 0.9, G4NN >0.5, suggested the high likelihood of G4 formation. The Us in the rG4 sequences are replaced by Ts in the comparative sequence analysis and G4 prediction.

Figure 3.

MALAT1 rG4-NONO protein interactions in vitro and in nuclear lysate. (A) EMSA with recombinant NONO (53–312) and MALAT1 rG4_04. (B) EMSA with recombinant NONO (53–312) and MALAT1 rG4_09. (C) EMSA with recombinant NONO (53–312) and MALAT1 rG4_04 MUT. (D) EMSA with recombinant NONO (53–312) and scramble G-rich sequence. (E) Binding curves of recombinant NONO (53–312) and MALAT1 rG4_02, MALAT1 rG4_04, MALAT1 rG4_05, MALAT1 rG4_06, MALAT1 rG4_09, MALAT1 rG4_10, MALAT1 rG4_04 MUT and scramble G-rich sequence. The bindings of NONO to rG4s are stronger than rG4 mutant and scramble G-rich sequence, suggesting the binding is rG4-specific. (F) rG4 pull-down assay with HEK293T nuclear lysate. Western blot result of rG4 pulldown shows that NONO is enriched by MALAT1_rG4_04 and MALAT1_rG4_09 wildtype rG4s but not rG4 mutants, and the interaction can be disrupted by adding 20 μM PDS. In each blot, the rG4 mutant with no PDS treatment is normalized to one. Three independent experiments are performed. Error bars represent the standard error of the mean (S.E.M.). The representative blot is shown here.

Figure 4.

MALAT1 rG4-NONO protein interaction in cells. (A) Scheme for the ChIRP-WB and qPCR. RNA binding protein (RBP) is crosslinked to lncRNA of interest in cells, followed by sonication. Biotinylated tiling probes are then hybridized to target lncRNA, and the complexes are purified using magnetic streptavidin beads, followed by stringent washes. The enriched lncRNA and RBP are isolated and processed by western blot and qPCR separately. (B) NONO protein is enriched by MALAT1 ChIRP, and their interaction can be disrupted by 10 μM G4 ligand PDS, indicating the binding is mediated by rG4. GAPDH is used as negative control in this experiment. Three independent experiments are performed. (C) MALAT1 RNA is pulled down by biotinylated probes, and PDS treatment do not have much effect on the pull-down efficiency. Both sets of MALAT1 qPCR primers shows great MALAT1 lncRNA enrichment. Xist lncRNA is used as a negative control in this experiment and no enrichment is observed. Three independent experiments are performed. Error bars represent the S.E.M.

Figure 5.

Suppression of MALAT1 rG4-NONO interaction by multiple G4 targeting tools. (A) Schematic representation of MALAT1 rG4-NONO interaction that is interfered by various G4 targeting tools including small molecule, peptide, and L-aptamer. (B) Saturation plot of PDS for its inhibition of MALAT1 rG4-NONO interaction. Reaction mixture contains 50 nM FAM MALAT1 rG4_04, 200 nM NONO (53–312) and increasing concentrations of PDS (0.15–5000 nM). The IC₅₀ is found to 498.2 ± 94.5 nM. (C) Similar set up as (B) except RHAU53 is used. The IC₅₀ value is found to be 621.1 ± 95.3 nM. (D) Similar set up as (B) except L-Apt.4–1c was used. The IC₅₀ value is found to be 618.1 ± 110.7 nM. (E) Filter-binding result of PDS for its inhibition of MALAT1 rG4-NONO interaction. Reaction mixture contains 2 nM Biotin MALAT1 rG4_04, 150 nM NONO (53–312) and increasing concentrations of PDS (0.15–5000 nM). (F) Similar set up as (E) except L-Apt.4–1c is used.