Fig 2: Endogenous TRBP interacts with S6Ks in primary HDLEC. (A) Analysis of TRBP and S6K2 interaction by PLA (red) in HDLECs growing in full growth factor media. Nuclei (blue) and F-actin (green) staining are also shown (scale bar: 10 μm). PLA events (in red) are indicated with yellow arrows. (B) Analysis of TRBP and DICER interaction by PLA (red) in HDLECs growing in full growth factor media. PLA events (in red) are indicated with yellow arrows. (C) Histogram of TRBP/S6K2 PLA events per cell detected in HDLECs cultured in full growth factor media. (D) Histogram of TRBP/S6K1 PLA events per cell detected in HDLECs cultured in full growth factor media.

Fig 3: UMD regulation requires miRNAs. a The mean changes in expression (log2 scale) of dEJ-free mRNAs embedding 7-mer target sites of random controls (gray), the 10 (blue), 30 (purple), and 50 (red) most abundant miRNA families under siUPF1-treated conditions are shown. The P value was calculated by comparing the mean change in the expression of the targets to those in the 1000 random control cohorts (see “Generation of random controls and statistical tests”) using a one sample t test. The same analyses were performed for our data (left), Wang et al. 2014’s data (middle), and Hurt et al.’s data (right). The error bars indicate the s.e.m. in the changes for the targets. b HeLa cells were transfected with siUPF1, siDicer1, or both siUPF1 and siDicer1. The downregulation of endogenous UPF1 and/or Dicer1 was confirmed by western blotting. Calnexin served as a loading control. c CDF graphs of changes in the expression of dEJ-free mRNAs between siUPF-treated and siControl-treated cells under Dicer1-depleted conditions are shown across different 3′UTR length bins. Otherwise, as in Fig. 1b–d. d Mean changes in the expression of dEJ-free mRNAs with 7-mer target sites between siUPF-treated and siControl-treated cells under Dicer1-depleted conditions. Otherwise, as in a. e Mean numbers of 7-mer miRNA target sites in the 3′UTRs of dEJ-free mRNAs are shown across different 3′UTR length bins. The sites were detected in the major 3′UTR isoform, as evident by our previous 3P-seq data profiled from HeLa (green) and mES (orange) cells11. f Mean changes in expression of dEJ-free mRNAs in siUPF1-treated cells against siControl-treated cells are shown across the different 7-mer site numbers of the ten most abundant miRNA families (red) and different random 7-mer sites (gray). Asterisks denote groups whose number of sites is significantly different from that of the controls (***P < 0.001; K–S test). g Mean changes in expression between dEJ-free mRNAs of siUPF-treated and siControl-treated cells under Dicer1-depleted conditions are shown for the different 7-mer site numbers. Otherwise, as in f

Fig 4: Chromatin immunoprecipitation assay of the binding of GABPA to the DICER1 promoter. (A) Gel electrophoresis showing DNA bands from DICER1 promoter PCR product. Lane 1 shows band from 2% input DNA, lane 2 shows band from pulled down DNA using GABPA antibody and lane 3 shows PCR product from cross-linked chromatin DNA incubated with normal rabbit IgG. (B) Chromatogram from the GABPA antibody pulled down DNA and subsequent amplification and sequencing of the DICER1 promoter using the reverse primer as sequencing primer. The sequence was blasted with a 100% match in the DICER1 promoter. A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0446.

Fig 5: let-7b-mediated DICER regulation causes global miRNA changes. (A) Relative mean expression of endogenous let-7b-5p miRNA assayed by TaqMan-based qPCR across the breast cancer progression panel. let-7b levels were first normalized to a small nucleolar RNA, RNU44. The Ct values were converted to fold change using the delta Ct method and setting the lowest gene expression (MCF10A.P cells, in this case) as 1. (B) Relative mean expression of endogenous DICER1 mRNA assayed by qPCR in MCF10.AT1 cells transfected with either scramble mimic or let-7b-5p mimics. DICER1 mRNA levels were first normalized to a housekeeping gene, RPL19. The Ct values in let-7b-5p mimic-treated cells were converted to fold change using the delta Ct method and setting the gene expression in scramble-treated MCF10.AT1 cells as 1. (C) Western blot analysis and its quantification showing repression in endogenous DICER1 protein caused by let-7b-5p mimics compared to scramble control mimics in preneoplastic MCF10.AT1 cells. DICER protein levels were normalized to a loading control, vinculin. The graphs represent mean values + SEM. (D) Relative mean expression of indicated miRNAs as assayed by qPCR in MCF10.AT1 cells transfected with either scramble mimic or let-7b-5p mimics. miRNA levels were first normalized to a loading control, RNU44. The Ct values in let-7b-5p mimic-treated cells were converted to fold change using the delta Ct method and setting the gene expression in scramble-treated MCF10.AT1 cells as 1. The graphs represent mean values + SEM. p values were calculated using student’s t test. * indicates p < 0.05.