Fig. 5: The NEXT complex mediates Xist RNA turnover in the cell nucleus.
From: m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics
a, Top, strategy showing in-frame insertion of FKBP12F36V into ZCCHC8. Bottom, western blot showing the protein level of ZCCHC8–FKBP12F36V after 0 or 2 h of dTAG-13 treatment. TBP was used as a loading control. The red and black arrows indicate the protein size for ZCCHC8–FKBP12F36V and untagged ZCCHC8. b, As in a but for ZFC3H1. METTL3 was used as a loading control. c, Box plot showing the allelic ratio of X-linked genes from ChrRNA-seq analysis for ZCCHC8 dTAG degron samples. The experimental design is as described in Fig. 1c. The red dashed line indicates the allelic ratio at 0.5. Samples (two independent clones, Z4F and Z12H) and conditions are indicated above and below, respectively. P values, as indicated, were calculated using a two-sided paired t-test. d, As in c but for ZFC3H1. Note that only the proximal 138 MB of X chromosome in the 11A clone is informative for allelic-speific analysis. In box plots (c,d), center lines indicate the median, box limits indicate the first and third quartiles and whiskers indicate 1.5× the IQR. e, Bar plot showing the expression level of Xist from ChrRNA-seq analysis for samples and conditions described in c. f, As in e but for ZFC3H1 clones described in d. Each bar in e,f represents one biological replicate. g, Schematic depicting alternative models for m6A-mediated regulation of Xist RNA turnover as discussed in the main text. m6A sites are indicated as lollipops with open triangle.
