Fig. 6: M1-Ubi of AGO2 interferes its recruiting miRNA-targeted mRNAs.

a, b Scatter plots of miRNA expression profiles by miRNA-Seq in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L (a) and HeLa cells knocking down HOIP (b). c, d M1-Ubi of AGO2 inhibited recruitments of mRNAs. Cumulative fraction analyses for mRNA transcripts recruiting to AGO2 were conducted by using RIP-Seq data in stable HeLa-Flag-AGO2 cells expressing HOIP and HOIL-1L (c, n = 7014 mRNA transcripts) and HeLa cells knocking down HOIP (d, n = 5788 mRNA transcripts). e, f Venn diagram analysis (e) and scatter plots analysis of mRNA transcripts recruited to AGO2 with more than 1.5-fold changes (f). g, h Several mRNAs bound to AGO2 were examined by RIP/qRT-PCR in above stable HeLa cells. i, j Cumulative fraction analyses for top 10 non-difference miRNA-targeted mRNA transcripts recruiting to AGO2 (i, n = 2792 mRNA transcripts; j, n = 982 mRNA transcripts) were conducted by using RIP-Seq data in stable HeLa cells. k LUBAC inhibit the association of let-7-targeted HMGA2 and c-MYC mRNAs with AGO2. HMGA2 and c-MYC mRNAs bound to AGO2 were measured by RIP/qRT-PCR. The RIP efficiency were determined by WB and Northern blotting analysis. l–n Knockdown of HOIP augmented the associations of let-7a-targeted mRNAs to AGO2. HeLa-shHOIP cells transfected with indicated plasmids were used for target mRNA pull-down assay (l) and GST-MS2 pull-down assay (m, n). l RNA bound fraction (beads) and unbound fraction (supernatant) were detected by WB. m A schematic illustration of the GST-MS2 pull-down assay. n AGO2 associated RNAs through let-7a were assessed by GST-MS2 pull-down. o K820R of AGO2 augmented its recruiting with let-7-targeted mRNAs. The abundance of let-7a and let-7a targeted HMGA2/c-MYC mRNAs associated with AGO2 or mutants were determined by RIP/northern blotting and RIP/qRT-PCR, respectively. qRT-PCR data were mean ± s.d., n > = 3 biologically independent samples, and P-values were determined by unpaired two-sided t-test (g, h, k, o). In box plots, the lines represent the median, first and third quartiles, the whiskers denote the minima and maxima; P-values were calculated using a two-sided Mann–Whitney U test for cumulative fraction analysis (c, d, i, j).