Fig. 5: HOXA-AS3 and HOXA6 mRNAs form a duplex RNA-RNA structure at their mutually overlapping regions.
From: Increased levels of the long noncoding RNA, HOXA-AS3, promote proliferation of A549 cells
a Genomic sequences of HOXA-AS3. Arrows show the direction of the transcription. b RNAfold and RNAcofold predict the binding capacity and the binding free energy, respectively, of HOXA-AS3 with its neighboring genes. c Expression of HOXA3, HOXA5, and HOXA6 quantified by qRT-PCR after HOXA-AS3 knockdown in A549 cells. d Western blot analyses of the protein expression levels of HOXA3, HOXA5, and HOXA6 after HOXA-AS3 knockdown in A549 cells. e The stability of HOXA6 mRNA over time was measured by qRT-PCR relative to time 0 after 5 μg/ml actinomycin (d) treatment in A549 cells transiently expressing HOXA-AS3 siRNAs or control siRNA. f The start and end positions of genes as indicated on the UCSC site (Genome Bioinformatics). The black arrows indicate the transcription direction. The schema in scale “+” at both sides of the strand represent the positive strand, and “−” represent the negative strand. Blocks with colors (green, blue, and red) represent exons. The red block in the HOXA-AS3 transcript represents the overlapping region (OL). The red block in HOXA6 mRNA represents the first exon and the OL region. “e” followed by a number denotes the exon and its serial number. The gray block represents the intronic portion of HOXA-AS3. The dotted filled lines between colored blocks represent introns in HOXA6. g Recombinase polymerase amplification (RPA) performed on RNA samples from A549 cells. Depicted are qRT-PCR results from two sets of primers and probes covering overlapping and nonoverlapping regions of HOXA6 mRNA. h Determination of the binding affinity of HOXA6 to HOXA-AS3 mRNA by microscale thermophoresis. The concentration of labeled HOXA-AS3 molecules was constant, whereas the concentrations of the nonlabeled binding partner HOXA6 molecules varied from 10 nM to 400 μM. NC, negative control; siRNA/HOXA-AS3, small interfering RNA for HOXA-AS3. *P < 0.05, **P < 0.01, and ***P < 0.001. All values are expressed as the mean ± SEM
