Fig. 8: DHX36 promotes Gnai2 translation by resolving the 5’ UTR rG4.

a Gnai2 protein and mRNA were detected in Ctrl and iKO SCs cultured for 48 h. b Gnai2 protein and mRNA were detected in proliferating Ctrl and Dhx36-KO C2C12 cells. c GNAI2 protein was detected in Ctrl, KO, or KO C2C12 cells treated with proteasome inhibitor MG132 for 8 h. d Left: SCs were treated with 2.5 or 5 μM cPDS and GNAI2 protein levels were detected by western blot. The relative intensity of each GNAI2 protein band normalized by α-Tubulin was calculated by ImageJ as the intensity for untreated cells set as 1. Right: Gnai2 mRNA was detected by qRT-PCR in the above cells. Gapdh mRNA was used as a normalization control. e Left: rG4 formation in Ctrl and iKO SCs was detected by QUMA-1 staining. Cells treated with RNase A were used as negative controls. Scale bar = 5 μm. Right: Boxplot of normalized rG4 fluorescence signals which were calculated from the indicated number of cells from three pairs of Ctrl/iKO mice by Matlab R2014b using in-house scripts. The average intensity from Ctrl cells was set as 1. Horizontal lines represent median values. Error bars show the distribution from the minimum to the maximum data points. The boxes extend from the 25th to 75th percentiles. Significance was calculated by Student’s t test (two-tailed unpaired), P = 3.1E-17. f EGFP reporter plasmids harboring WT or Mut full-length 5’ UTR, rG4#1, or rG4#2 were transfected into C2C12 cells and EGFP protein was detected by western blot. α-Tubulin was used as the internal loading control. g EGFP mRNA was detected by qRT-PCR from the above samples. h EGFP reporter plasmids harboring WT full-length 5’ UTR, rG4#1, or rG4#2 were transfected into Ctrl or Dhx36-KO C2C12 cells and EGFP protein was detected by western blot with α-Tubulin used as the internal loading control. i EGFP mRNA was detected by qRT-PCR from the above samples. Data represent the average of three independent experiments ± s.d. in a, b, d, g, and i. Source data are provided as a Source Data file.