Figure 5: MEG3 lncRNA regulates its target genes through triplex structure formation.

(a) Predicted GA-rich motifs enriched in all MEG3 peaks and peaks associated with deregulated genes using MEME-ChIP. (b) Number of TrTS over the MEG3 peak summits and neighbouring regions, predicted by Triplexator⁴¹. (c) The schematic shows the MEG3 TFO used in the triplex assays. The exons are colour-coded as described before. (d) Electrophoretic mobility shift assay. End-labelled dsDNA oligos (sequences provided in the schematic with gene name) were incubated alone (lane 1) or with increasing concentrations of MEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) or with increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNA oligos were incubated with MEG3 TFO and treated with either RNase A (lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3 ssRNA TFO in the presence of either unlabelled specific competitor (lane 6) or nonspecific competitor (lane 7). (e) TGFBR1-associated MEG3 peak sequence and its mutated version (the changed nucleotides are in red) were incubated alone (lanes 1 and 7) or with MEG3 TFO. Arrow indicates complex formation. (f,g) Enrichment of MEG3 peak sequences using biotin- and psoralen-labelled MEG3 TFO. RNase H-treated lysates were used to capture the labelled MEG3 TFO using streptavidin beads. The enrichment of MEG3 peaks is presented as the ratio between MEG3 TFO and control oligo (±s.d., n=3). (h) RT–qPCR analysis of gene expression in BT-549 cells transfected with either MEG3 TFO or control RNA oligo. Expression in MEG3 TFO presented relative to the control oligo (±s.d., n=3). *P<0.05, Student’s t-test (two-tailed, two-sample unequal variance). (i) Left panel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO (ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA are shown in red. Right panel: the sum of the individual CD spectra for TGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, and the sum of the individual CD spectra for TGFB2 dsDNA and the control ssRNA is shown in red. Inset in the left and right panel shows the difference between the two spectra.