Fig 1: “Quencherless” fluorogenic substrates allow real-time monitoring of enzymatic activity of DICER and reconstituted RISC complex for enzyme kinetics assays.Recombinant human RNAi proteins were expressed in insect cells, purified and analyzed by SDS-PAGE (2.5 µg/lane) with Coomassie Blue staining (A). Lanes: SeeBlue Plus2 pre-stained markers (M), DICER (1), TRBP (2) and AGO2 (3). Continuous enzymatic assays of DICER using fluorogenic substrates (250 nM) bearing asymmetric overhangs (3’-dinucleotide overhang on anti-sense strand and long sense overhang) are shown (B), and linearity with enzyme concentration is shown (C). Continuous assays of reconstituted human RISC (combinations of purified enzymes) using DICER substrate BoGD664 (250 nM; D-E) or fluorogenic siRNA (AGO-loading substrate BoPsi664; 250 nM; F). AGO2 increases enzymatic activity of reconstituted RISC: (DICER+AGO2 > DICER alone (E-F). AGO2 or TRBP alone show no activity (E-F). *, p<0.05.
Fig 2: Binding interactions in the RISC complex are functionally determined using enzyme kinetics.DICER-AGO2 binding interaction was assessed by enzymatic activity assays modeled using the Morrison equation [17]. Titration of DICER (25 nM) with AGO2 enhances enzymatic activity for cleavage of either fluorogenic DICER substrate (250 nM; A) and the fluorogenic siRNA (AGO-loading substrate; 250 nM; B) apparently via high-affinity binding interaction. AGO loading is dependent on AGO2 concentration and requires DICER (B). Michaelis-Menten kinetics were observed for DICER and minimal reconstituted RISC using both DICER substrates (C-D). AGO-loading siRNA exhibited kinetics consistent with a substrate inhibition model (E), whereas DICER or DICER+TRBP demonstrate minimal enzymatic activity. Lesser apparent fluorogenic activity was observed in combination with the dsRNA-binding protein TRBP.
Fig 3: FAM172A influences the nuclear localization of AGO2, but not AGO1.(A) Immunofluorescence analysis of AGO2 distribution in WT and Fam172Tp/Tp e10.5 MEFs at high density (200,000 cells/cm2), with the nuclei stained using DAPI. (A, B) Quantification of relative fluorescence intensity for anti-AGO2 staining in the nucleus and cytoplasm (N:C ratio, expressed in log 2 scale) using images such as those displayed in (A). Yellow and white dashed lines in zoomed-in views in A delineate areas of measurement for the nucleus and cytoplasm, respectively. (C) Western blot analysis of AGO2 protein levels in nuclear and cytoplasmic fractions of WT and Fam172aTp/Tp e10.5 MEFs grown at high density (200,000 cells/cm2) showing a specific decrease in the nucleus of Fam172aTp/Tp cells (N = 3). Lamin A/C and GAPDH are used as loading control for nuclear and cytoplasmic fractions, respectively. (C, D) Quantitative analysis of relative anti-AGO2 signals in mutant versus WT cells after normalization with relevant loading control (GAPDH for total and cytoplasmic fractions; Lamin A/C for nuclear fraction), as determined via densitometry (ImageJ) using images such as those displayed in (C). (E) Immunofluorescence analysis of AGO1 distribution in WT and Fam172Tp/Tp e10.5 MEFs at high density (200,000 cells/cm2), with the nuclei stained using DAPI. (E, F) Quantification of relative fluorescence intensity for anti-AGO1 staining in the nucleus and cytoplasm (N:C ratio, expressed in log 2 scale) using images such as those displayed in (E). (G) Immunofluorescence analysis of FAM172A distribution in WT e10.5 MEFs transfected or not with FLAGAGO2 (N = 3). Red arrowheads compare fluorescence intensity in nuclei of transfected and non-transfected cells. (H) Western blot analysis of FAM172A isoforms in NIH 3T3 fibroblasts, Neuro2a (N2a) neuroblasts, and R1 embryonic stem cells (N = 3). Scale bar, 20 µm. N = number of biological replicates, n = number of cells. **P = 0.01, ****P = 0.0001; t-test.
Fig 4: AGO2 overexpression can functionally compensate for the loss of FAM172A ex vivo.(A, B) Quantitative analysis of cell proliferation (A) and alternative splicing of Cd44 (B) in e10.5 Fam172aTp/Tp MEFs, after co-transfection with a GFP expression vector and either MYCFAM172A (WT, E229Q-mutant or NLS-mutant) or FLAGAGO2 (WT, NLS-containing, and deletion mutants) expression vectors. Cell proliferation is expressed in % of GFP-positive cells based on double-immunofluorescence staining of GFP and the proliferation marker Ki67. Alternative splicing of Cd44 was analyzed by RT–qPCR after FACS-mediated recovery of GFP-positive cells. Expression levels of variable exons 8/9 (vE8/9) were normalized against levels of a constant exons 4/5 (cE4/5), and splicing fold change was then determined by comparison to the reference value (dashed line) obtained with WT control embryos (N = 5 per condition). (C) Immunofluorescence analysis of the distribution of NLS-containing FLAGAGO2 in transfected N2a cells, with nuclei stained using DAPI. Scale bar, 20 µm. *P = 0.05, **P = 0.01, ***P = 0.001 and ****P = 0.0001; t-test.
Fig 5: AGO2 nuclear import depends on the status of FAM172A phosphorylation.(A) 5-h-long time-lapse recordings of BiFC signal generated using N-VenusAGO2 and C-VenusF172A in N2a cells treated with leptomycin B or vehicle (ethanol) only, with or without 4,5,6,7-tetrabromobenzotriazole. Red arrowheads compare relative fluorescence intensity in the nucleus. (A, B) Quantification of relative BiFC signal intensity in the nucleus and cytoplasm (N:C ratio, expressed in log 2 scale) using images such as those displayed in (A). (C) 5-h-long time-lapse recordings of the BiFC signal generated using N-VenusAGO2 and C-VenusF172A (comparing phosphodead [P-] and phosphomimetic [P+] versions) in N2a cells treated with leptomycin B or vehicle (ethanol) only. Red arrowheads compare relative fluorescence intensity in the nucleus. (C, D) Quantification of relative BiFC signal intensity in the nucleus and cytoplasm (N:C ratio, expressed in log 2 scale) using images such as those displayed in (C). Data for C-VenusF172A[WT] are the same as initially displayed in Fig 3F (both assays were performed at the same time), being duplicated here for comparison purposes only. (E) Comparison of overall BiFC fluorescence intensity between N-VenusAGO2–C-VenusF172A[WT], N-VenusAGO2–C-VenusF172A[P-], and N-VenusAGO2–C-VenusF172A[P+]. Mean fluorescence intensity was determined using flow cytometry 24 h after transfection in N2a cells. (F) Immunofluorescence analysis of the distribution of MYC-tagged WT, phosphodead (P-), and phosphomimetic (P+) versions of murine FAM172A protein in transfected N2a cells, with nuclei stained using DAPI. (F, G) Quantification of relative fluorescence intensity of anti-MYC staining in the nucleus and cytoplasm (N:C ratio, expressed in the log 2 scale) using images such as those displayed in (F). Data for the MYCF172A[WT] condition are the same as initially displayed in Fig 3D (both assays were performed at the same time), being duplicated here for comparison purposes only. (A, C, F) Scale bar, 20 µm (A, C) and 10 µm (F). N = number of biological replicates, n = number of cells. *P = 0.05, **P = 0.01, ***P = 0.001, and ****P = 0.0001; t-test. (Further data can be found in Fig S5).
Supplier Page from Sino Biological, Inc. for Human AGO2 / Argonaute 2 / EIF2C2 Protein (His Tag)