Fig. 4: Loss of miR106a-RepA partnering compromises the assembly of Wtap and m⁶A formation on Xist.

A Volcano plot depicting differential pull-down of 62 Xist-interacting proteins in control and miR106a-depleted cells. Blue indicates proteins downregulated (n = 4), Red indicates proteins upregulated (n = 2), and Gray indicates no change (n = 56) in miR106a-depleted cells relative to control cells. n = 3. B RIP assay monitoring binding of Wtap to Xist and Actin in control or LNAs-treated cells. n = 3. *p = 0.015. C Immunoblot of proteins retrieved by Xist and isogenic control probes from H4SV cells expressing NS or miR106sp by ChIRP. Input is 2% of the total protein. The representative images (Left) and the results quantified from three experiments (Right) are shown. Wtap: Control vs. miR106sp, *p = 0.032; Mettl3: Control vs. miR106sp, *p = 0.023; Mettl14: Control vs. miR106sp, **p = 0.0029. D qRT-PCR analysis monitoring m⁶A, m⁵C, and Ψ enrichment on Xist in H4SV cells expressing control or miR106sp. The predicted non-binding site (NBS) in Xist for m⁶A, m⁵C, and Ψ is used as a negative control. n = 3. For m⁶A: NS vs. miR106sp, **p = 0.0069. E Scheme of in vitro methylation assay with RepA using ³H-labeled SAM (Left). Slot blot assay monitoring methylation of in vitro synthesized RepA incubated with whole cell lysate prepared from cells expressing NS or miR106sp in a time-dependent manner. The representative images (Middle) and the results quantified from three experiments (Right) are shown. RepA pre-treated with RNase was used as a negative control. For NS: 4 h, **p = 0.0024; 8 h, **p = 0.0048; 24 h, **p = 0.0054. For miR106sp: 4 h, *p = 0.027; 8 h, **p = 0.0039; 24 h, **p = 0.01. F RIP assay monitoring binding of Ythdc1 to Xist and Gapdh in H4SV cells ectopically expressing NS or miR106sp or Wtap shRNA. n = 3. Control vs. miR106sp, *p = 0.035; Control vs. Wtap KD, *p = 0.049; Control vs. miR106sp+Wtap KD, *p = 0.033; miR106sp vs. miR106sp+Wtap KD, *p = 0.05; Wtap KD vs. miR106sp+Wtap KD, **p = 0.0037. G qRT-PCR analysis of pSM33 Xist-(BoxB)₃ ES cells expressing DC1 and Wtap shRNA or miR106sp on Xist-mediated gene silencing. Gene expression was normalized to Gapdh and Gpc4 levels prior to Xist induction in cells expressing empty vectors. n = 3. For WTAP KD, Dox^- vs. Dox⁺, *p = 0.012, Dox⁺ vs. Ythdc1⁺, *p = 0.014, dox^- vs. Ythdc1⁺, ***p = 0.00029; For miR106sp, Dox^- vs. Dox⁺, *p = 0.03, Dox⁺ vs. Ythdc1⁺, **p = 0.0029, dox^- vs. Ythdc1⁺, **p = 0.0045. H Schematic model of miR106a-directed regulation of X_i silencing. Data is expressed as mean ± SD (B–G). Unpaired, two-sided t-test with no multiple adjustments (B–G).