Fig. 2: miR106a physically interacts with RepA in Xist RNA.

A Strategy to capture miR106a-RepA complex (Left). Competitive elution of RepA from miR106a-RepA complex using mismatch, perfect, or imperfect complementary capture oligonucleotides. Samples pretreated with RNase were used as a negative control. The experiment was performed three times; a representative image from one experiment is shown (Middle), and the results are quantified (Right) from three different experiments. For transcript A, Oligo 2, *p = 0.013, Oligo 3, *p = 0.034, Perfect match ^nsp = 0.22; For transcript B, Oligo 4, ^nsp = 0.061, Oligo 5, ^nsp = 0.17, Perfect match ^*p = 0.031; For transcript C, Oligo 1, **p = 0.0034, Oligo 6, ^nsp = 0.083, Perfect match ^nsp = 0.126. B Outline of the modified PARIS2 method. C PARIS2 assay validates predicted miR106a interactions (Colored arcs) in the RepA region. Fractions of duplex groups (DGs) corresponding to each site are shown. Numbers in parentheses are the counts of gapped alignments in each DG. n = 2. D The percentage of mapped reads for each amplicon from the replicates is shown. Data were analyzed from two experiments and expressed as mean ± SD. E qRT-PCR analysis following PARIS2 assay monitoring the enrichment of miR106a-RepA duplex in control and LNA-treated cells. The predicted miR106a non-binding region (NBS) in Xist is a negative control. n = 3. For MRE 1, **p = 0.0072; MRE2, **p = 0.0056; MRE3, **p = 0.0043; MRE4, *p = 0.020. F RIP assay monitoring miR106a-Xist interaction in H4SV cells using biotinylated miR106a (Left) and Xist-specific probes (Right). The predicted miR106a non-binding region (NBS) in Xist and U6 is a negative control. n = 3. For miR106sp RIP, NBS, *p = 0.029; Gapdh, *p = 0.03. For Xist RIP, *p = 0.015. Data is expressed as mean ± SD (A, E, F). Unpaired, two-sided t-test with no multiple adjustments (A, E, F).