Germline AGO2 mutations impair RNA interference and human neurological development

Abstract

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.

Fig. 1. Location of identified AGO2 germline mutations.

a Domain structure of AGO2 and position of the single amino acid mutations using the structure of human AGO2 in complex with a miRNA and a target RNA. Guide and target RNA are depicted in orange and red, respectively. The recurring mutations are designated in brackets. b Genomic region, chr8.hg19:g.(141,582,269-141,817,600)del, of the 235.3-kb deletion identified in case 21, involving the first three AGO2 exons and the last 23 PTK2 exons.

Fig. 2. Germline AGO2 mutations impair shRNA-mediated silencing.

a–c AGO2-deficient HEK293T cells were transfected with mRFP-Shank3 (a), mRFP-DDX1 (b), or GFP-tagged δ-catenin (c) along with shRNA constructs targeting Shank3 (a; in pLVTHM vector coexpressing GFP), DDX1 (b; in pSuper) or δ-catenin (c; also in pSuper), and Flag/HA-tagged (Shank3, δ-catenin) or GFP-tagged (for DDX1) AGO2 variants as indicated. Efficient transfection (>70 % of cells) was verified by fluorescence microscopy using appropriate fluorophores (GFP in (a) and (b); RFP in (c). Cells were lysed and lysates were analysed by Western Blotting using the antibodies indicated. In each case, each experiment was repeated eight times with similar results. d Quantification of the representative immunoblots shown in (a–c). The expression levels are relative to those in control cells without an AGO2 construct (first lane in each immunoblot). e Quantification of a second set of AGO2 mutants. For (d, e), data are means + SEM. Data for mutant and control conditions were compared to wt. *, **, ***p < 0.05, 0.01, 0.001, respectively; n = 8 biologically independent experiments in (d); one-way-ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 6–14 biologically independent experiments in e for Shank3; mixed-effects analysis, followed by Holm-Sidak’ multiple comparisons test; n = 6 biologically independent experiments in (e) for δ-catenin; one-way ANOVA, followed by Dunnett’s multiple comparisons test. f Knockdown of DDX1 with DDX1 shRNA was performed as in (b), but using F/H-tagged AGO2 in combination with GFP, or in combination with GFP-tagged AGO2 variants. Note that the GFP-tagged AGO2 mutants do not interfere with the knockdown capacity of F/H-AGO2 WT. n = 8 biologically independent experiments; data are means +/− SD; ****p < 0.0001; one-way ANOVA, followed by Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.

Fig. 3. Reduced target dissociation of AGO2 germline mutants.

a 293 cells were transfected with F/H-tagged AGO2. After immunoprecipitation, phosphorylation of S824 alone, of the S824–S834 cluster and of S387 was measured by a targeted quantitative mass spectrometry approach (Selected reaction monitoring with isotopically labeled spike-in peptides). The y-axis represents the percentage of individual phosphorylated peptide species assuming the sum of singly, multiply and non-phosphorylated peptides to be 100%. Significance was assessed by two-sided Student’s t test in relation to WT. n = 3 biologically independent experiments. Data are presented as mean +  SD. *, **, ***p < 0.05, 0.01, 0.001, respectively. b RNA was isolated from F/H–AGO2 immunoprecipitates and analyzed by qRT-PCR using primers for the genes indicated. The significance was assessed by two-sided Student’s t test in relation to WT. n = 3 biologically independent experiments. Data are presented as mean +  SD. *, **, ***p < 0.05, 0.01, 0.001, respectively. Source data are provided as a Source Data file.

Fig. 4. Molecular dynamic simulation of effects of AGO2 mutations.

a Distance between the Cα atom of I365 and the glycosidic N atom of the g7 residue of the guide (I365α-g7(N)). Populations of all core-RISC trajectories were normalized to the same bin number (60). Black and orange dashed vertical lines denote the reference X-ray structures of the int and nonint core-RISC states (4OLA and 4W5N, respectively). The red dashed line denotes the X-ray structure of the helix-7 mutant (5WEA). b Distance between the c.o.m. of the 3′-end of the guide and the c.o.m. of the PAZ domain (g21-PAZ) along the trajectories of all RNA-bound states. Note that the relatively short length of the individual MD trajectories could affect the results. Color code of the states in both a and b panels: apo-Ago2— green, int core-RISC—gray, nonint core-RISC—orange, g2-8 holo-RISC—magenta and g2-7 holo-RISC—blue. cd motion of the helix7 and the PAZ domain along the open-closed mode (left panel, gray scale histogram). The histograms are calculated by concatenating the last 100 ns of non-biased trajectory of each variant with mismatched RNA duplex. Colored circles depict the maximum population density of each trajectory. Black cross denotes the maximum population density of the WT AGO2 with guide RNA. Right panel: population histograms on α7-MID corresponding to the maxima on the left panel. Equivalent analysis of the variants in in complex with a fully matched seed duplex is shown on Fig. S16.

Fig. 5. AGO2 germline mutations lead to an increased number of dendritic P-bodies.

a Primary cultured murine hippocampal neurons were transfected with GFP control or GFP-AGO2 WT and variants and Tomato-red. Staining for the dendritic marker MAP2 is shown in gray; inserts display the GFP signal in cell bodies. Boxed areas are magnified below for GFP- and MAP2 signals. Arrows indicate dendritic GFP-AGO2 clusters. Similar results were obtained in three independent biological experiments; results are quantified in (c, d, e). Scale bars: 20 μm in overview pictures; 5 μm in inserts. b Neurons expressing GFP-AGO2 were co-stained for Dcp1a. Similar costaining results were obtained in two biologically independent experiments for wt and F182del, L192P and M364T mutants, with 30 cells analyzed per experimental condition Scale bar: 5 μm. c–e Quantification of GFP-AGO2 clusters in cell bodies (c), GFP-AGO2 clusters in dendrites (d), and primary dendrites per cell (e). Data for mutant and control conditions were compared to wt. *, **p < 0.05, 0.01, respectively; Brown–Forsythe and Welch one-way-ANOVA, followed by Dunnett’s T3 multiple comparisons test; n = 3; mean ± SD). Source data are provided as a Source Data file.

Fig. 6. Global transcriptome alteration in primary fibroblasts of AGO2 patients.

a, b Venn diagram of upregulated (a) and downregulated (b) transcripts in fibroblasts isolated from AGO2 patients (cases 2, 3, and 14) compared to five age-matched controls. The number of the upregulated (a) or downregulated (b) genes in all three cases are marked red, whereas deregulated genes in both cases bearing the p.L192P mutation are marked yellow.