Fig 2: BGLF2 colocalizes with TNRC6 foci.HONE-1 cells were co-transfected with a plasmid expressing either HA-TNRC6A (upper panel) or HA-TNRC6B (lower panel) and either pCMV3FC-BGLF2 or pCMV3FC empty plasmid control (EP). Twenty-four hours later, cells were fixed and stained with DAPI, anti-FLAG and anti-HA antibodies. Scale bar = 10 µm.

Fig 3: Interactions of ss-siRNAs with RNAi factors AGO2 and GW182 in GM06151 patient-derived fibroblasts. (A) Biotin-labeled ss-siRNA ISIS 580940 inhibits ATX-3 expression. (B) Western blot image showing pull-down of AGO2 after incubation with biotinylated ISIS 580940 and a biotin:strepavidin purification. (C) Effect of reducing AGO1~4 expression to the function of ss-siRNA 537775 on ATX-3 expression. Anti-AGO siRNAs and negative control siRNA CM were transfected first in SCA3 patient fibroblasts. After 3 days, ss-siRNA 537775 was added. (D and E). RIP assay examining the involvement of AGO2 or GW182 in ss-siRNA directed mRNA targeting. SCA3 patient fibroblast cells (GM06151) were treated with 25 nM of ss-siRNAs 537775 and a negative control. RNA was isolated from immunoprecipitation of anti-AGO2 or anti-GW182 antibodies. The levels of AGO2 or GW182-associated ATXN3 mRNA were quantified by qPCR. CM: non-complementary duplex RNA. Statistic significance was calculated by t-test. **P < 0.01, *P < 0.05 relative to negative control. For (D and E) the negative control was an ss-siRNA with a sequence unrelated to ATX-3.

Fig 4: Overexpression of Ago proteins inhibits the nuclear transport of the dotted foci of GFP-signals of myc-GFP-TNRC6A-NES-mut proteins. (A–D) HeLa cells expressing myc-GFP-TNRC6A-NES-mut with either of the indicated proteins, FLAG/HA-FL (A), -Ago1 (B), -Ago2 (C), or -Ago3 (D), were stained with an anti-HA antibody, followed by Cy5-conjugated anti-mouse IgG. Fluorescent images are shown from the left: GFP signals of myc-GFP-TNRC6A-NES-mut; Cy5 signals of the second antibody against anti-HA antibody; the merged images of GFP and Cy5, in which GFP is shown in green, the Cy5 in magenta; the merged images with DAPI (blue). Bars, 20 µm. (E) The ratio of cells expressing the dotted GFP-signals of myc-GFP-TNRC6A-NES-mut exclusively in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow). (F) Western blot of myc-GFP-TNRC6A-NES-mut and control FLAG/HA-FL, or FLAG/HA-tagged Ago1, Ago2, and Ago3 proteins. An anti-tubulin antibody was used as a loading control.

Fig 5: T6B fusion protein prevents miRNA-induced silencing complex (miRISC) assembly and impairs microRNA (miRNA) activity in vitro.(A) Schematics of T6B action: T6B competes with TNRC6 for binding to AGO proteins preventing miRISC assembly. (B) Schematics of the size-exclusion chromatography (SEC) assay for the fractionation of AGO-containing complexes according to their molecular weight. (C) SEC profiling of miRISC components upon T6B expression: total lysates from HCT116 cells expressing no fusion protein (upper panel), T6B (middle panel), or T6BMut (lower panel) were fractionated as described in (B) and immunoblotted to detect AGO2, TNRC6A, T6B, and PABP1. For each blot, the relative signal intensity was assessed by densitometric analysis. (D) RNAseq analysis of total and small RNAs isolated from mouse embryo fibroblasts (MEFs) cell lines expressing either no fusion protein, T6B, or T6BMut (n = 3 for each cell line). Upper panel: bubble plot of target de-repression against miRNA abundance. The mean log2-fold change (T6B or T6BMut vs. control) of predicted targets for each conserved miRNA family was calculated, converted to a z-score and is plotted on the x-axis against the miRNA family abundance (log of the sum of read counts for each member of the family). The size of each circle is proportional to the number of predicted targets. A positive z-score indicates that the targets for that family are preferentially upregulated upon T6B expression, while a negative score would indicate preferential downregulation. Expression of T6B, but not of T6BMut, causes preferential upregulation of miRNA targets of the most miRNA families and the effect is roughly proportional to each miRNA family abundance. Lower panel: cumulative distribution plot of predicted let-7 targets compared to background in T6B-expressing MEFs. (E) Scatter plots of miRNA abundance as determined by small-RNAseq of total RNA extracted from MEFs expressing either T6B or T6BMut (n = 3 for each cell line). Each dot represents a miRNA in miRbase. (F) Effect of T6B expression on AGO2 slicing activity. MEFs expressing either T6B or T6BMut were transfected with siRNAs targeting GAPDH mRNA (siGAPDH) or with scramble siRNA (siCTL). Levels of GAPDH, T6B, and tubulin were assessed by immunoblot 72 hr post-transfection. T6B and T6BMut have slightly different migration on PAGE, as previously observed by Hauptmann et al., 2015. Figure 1—source data 1.RNAseq, differential gene expression, mouse embryo fibroblasts. Figure 1—source data 2.z-scores and miRNA family abundance, mouse embryo fibroblasts. Figure 1—source data 3.Small RNAseq, microRNA counts, mouse embryo fibroblasts. Figure 1—source data 4.Unedited blots shown in Figure 1C. Figure 1—source data 5.Unedited blots shown in Figure 1F. Figure 1—source data 6.Unedited blots shown in Figure 1F. Figure 1—source data 7.Uncropped blots shown in Figure 1F.