Figure 4: AGO2 mediates transcriptional inhibition of GNG12-AS1.

(a) HB2 cells were transfected with siRNA to exon 1 or exon 7 of GNG12-AS1 together with control siRNA and siRNA targeting AGO2 (A2) (a) or AGO1 (A1) (Supplementary Fig. 9a). Expression levels of AGO2, AGO1, DIRAS3 and GNG12-AS1 were normalized to GAPDH and compared with control siRNA by qRT–PCR. AGO1 levels were not affected by either single AGO2 siRNA or double knockdown of AGO2 and GNG12-AS1. Transcriptional upregulation of DIRAS3 can be rescued by depletion of AGO2 and siRNA targeting exon 1 of GNG12-AS1, whereas siRNA-mediated reduction of GNG12-AS1 with both siRNAs expression can be rescued by depletion of AGO2. (b) AGO2 ChIP analysis in HB2 cells after siRNA depletion of GNG12-AS1. The x axis shows enrichment of AGO2 at regions described in Fig. 3a. AGO2 binding was enriched only at the TSS of GNG12-AS1 (a) after siRNA to exon 1. The GAPDH TSS was used as negative control region for AGO2 ChIP. (c) RIP from nuclear HB2 extracts showing association of AGO2 with GNG12-AS1 transcript after treatment of cells with exon 1 but not exon 7 siRNA. U1 and GAPDH were used as negative control RNAs for AGO2 RIP. RIP enrichments are presented as % of input RNA. For all the graphs, error bars indicate s.e.m. (n=3 biological replicates). *P<0.05 and **P<0.01 by two-tailed Student’s t-test.