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. 2024 Mar 5;121(10):e2308255121.
doi: 10.1073/pnas.2308255121. Epub 2024 Feb 27.

Modeling neurodevelopmental disorder-associated human AGO1 mutations in Caenorhabditis elegans Argonaute alg-1

Ye Duan #  1   2 , Li Li #  3 , Ganesh Prabhakar Panzade  3 , Amélie Piton  4 , Anna Zinovyeva  3 , Victor Ambros  1
Affiliations

Modeling neurodevelopmental disorder-associated human AGO1 mutations in Caenorhabditis elegans Argonaute alg-1

Ye Duan et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNA) associate with Argonaute (AGO) proteins and repress gene expression by base pairing to sequences in the 3' untranslated regions of target genes. De novo coding variants in the human AGO genes AGO1 and AGO2 cause neurodevelopmental disorders (NDD) with intellectual disability, referred to as Argonaute syndromes. Most of the altered amino acids are conserved between the miRNA-associated AGO in Homo sapiens and Caenorhabditis elegans, suggesting that the human mutations could disrupt conserved functions in miRNA biogenesis or activity. We genetically modeled four human AGO1 mutations in C. elegans by introducing identical mutations into the C. elegans AGO1 homologous gene, alg-1. These alg-1 NDD mutations cause phenotypes in C. elegans indicative of disrupted miRNA processing, miRISC (miRNA silencing complex) formation, and/or target repression. We show that the alg-1 NDD mutations are antimorphic, causing developmental and molecular phenotypes stronger than those of alg-1 null mutants, likely by sequestrating functional miRISC components into non-functional complexes. The alg-1 NDD mutations cause allele-specific disruptions in mature miRNA profiles, accompanied by perturbation of downstream gene expression, including altered translational efficiency and/or messenger RNA abundance. The perturbed genes include those with human orthologs whose dysfunction is associated with NDD. These cross-clade genetic studies illuminate fundamental AGO functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms.

Keywords: Argonaute; disease modeling; intellectual disability; microRNA; neurodevelopmental disorder.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
C. elegans alg-1 NDD mutants display alg-1 loss-of-function and antimorphic phenotypes. (A) Protein sequence alignment of the regions surrounding the amino acids corresponding to human AGO1 F180, G199, V254, and H751. Alignment includes human AGO1-4, C. elegans ALG-1 and ALG-2. The ALG-1 amino acid numbers indicated at the Bottom correspond to C. elegans ALG-1 isoform a (ALG-1a). (B) ALG-1 domain organization. The unstructured, non-conserved sequence at the N-terminus (aa1-187) of cel-ALG-1a is not shown. (C) AGO2::miRNA::target complex structure (PDB::6MFR) highlighting AGO1 F180, G199, V254, and H751 residues (20). Side chains of the above amino acids are presented as sticks. (D and E) Quantification of vulval defect phenotypes, represented by the lethality of young adult hermaphrodites (D) and reduction in the number of progeny per animal (E). Lethality is categorized as vulval integrity defect (lethality by bursting, Bst) or egg laying defect (lethality by matricide, Bag). (F) Quantification of vulva integrity defect of the alg-1 NDD mutations with alg-2(+) or alg-2(null) genetic backgrounds. (G) Representative fluorescent images of col-19::gfp expression patterns from WT (top) and mutants alg-1(G199S) (middle) or alg-1(H751L) (bottom) animals. (Scale bar, 25 µm.) (H) Quantification of col-19::gfp expression phenotypes. The statistical significance is analyzed by Fisher’s test (lethality and abnormal col-19:gfp expression) and Student t test (brood size).
Fig. 2.
Fig. 2.
The C749-S750-H751 subregion is functionally critical to ALG-1. (A) Visualization of the human AGO2 side-chains equivalent to human AGO1 C749, S750, and H751 in AGO2::miRNA::target complex (PDB:: 6MDZ) (20). Dashed lines and numbers indicate distances between adjacent atoms (Å). (B) Lethality, brood size, and abnormal col-19::gfp expression phenotypes scored at 25 °C.
Fig. 3.
Fig. 3.
The alg-1 NDD mutations cause allele-specific disruptions of miRNA expression and altered profiles of miRNAs associated with ALG-1. (A) Western blotting for ALG-1 protein in input and ALG-1 immunoprecipitated samples of wildtype, alg-1(null), and alg-1 NDD mutants. (B) Venn diagrams showing numbers of miRNAs with statistically significant up/down-regulated levels (Fold change > 2; FDR < 0.05). Results for alg-1(V254I) mutant are not shown because no significant perturbation was observed in ALG-1(IP), with a single miRNA up-regulated in alg-1(V254I) input (SI Appendix, Fig. S3 and Dataset S1). (C) Heatmap showing the levels of abundant (≥10 RPM) miRNAs in input and ALG-1 IP from wildtype and alg-1 NDD mutants. Data are shown as log2(RPM).
Fig. 4.
Fig. 4.
alg-1 NDD mutations lead to altered guide/passenger (miR/miR*) ratios. (A and B) Changes in miR/miR* ratio in input (B) and ALG-1 IP (C). log2FC miR*/miR ratio in wildtype versus mutant animals (Y-axis) is plotted against miRNA abundance in wildtype (X-axis). Burgundy dots represent miRNAs with reversed miRNA strand bias (miR* > miR), red dots represent miRNAs whose miR* strands were upregulated ≥twofold with P ≤ 0.05, and orange dots represent miRNAs whose miR* strands were upregulated ≥twofold but did not reach statistical significance. Dashed lines, |log2FC| = 1. (C) miRNA fold change comparison between input and ALG-1 IP. miRNAs with |FC| > 2 and P < 0.05 are color-coded to indicate miRNA up- or down- regulation in input and ALG-1 IP, and input or ALG-1 IP only. (D) miRNAs that exhibited reversed miRNA strand bias in input and/or ALG-1 IP. miRNA* are marked with an asterisk(*).
Fig. 5.
Fig. 5.
alg-1 NDD mutations lead to strong translatome perturbations in C. elegans. (A) Volcano plots of the ribosome protected fragments (RPF) detected in ribosome profiling of alg-1 NDD late L4 larvae. Colored dots represent perturbed genes with statistical significance (|log2FC| > 1.5, p.adj < 0.1). Also see Dataset S2. (B) Principal component analysis of translatomes of alg-1 NDD and alg-1 null. Identical color points indicate biological replication. (C) Venn diagram for the total perturbed genes in the alg-1 NDD mutants. (D) RPFs of heterochronic genes whose gain-of-function mutations cause heterochronic phenotypes and have been genetically confirmed to be C. elegans miRNA targets. (E) Visualization of set1-set3 antimorphic perturbed (amp) genes. For each gene, the log2FC of the null/WT is plotted on the x-axis, and the log2FC of alg-1 NDD mutant/WT is plotted on the y-axis. Solid dots indicate perturbed genes with statistical significance (|FC| > 1.5, p.adj < 0.1). See also Dataset S3. (F) Venn diagrams of set1-set3 amp genes in the alg-1 NDD mutants.
Fig. 6.
Fig. 6.
Antimorphic ALG-1 NDD miRISC sequester miRNAs into non-functional complexes, leading to a greater miRNA loss-of-function than in the absence of ALG-1. (A) Illustrative models of wild-type, alg-1(null) and ALG-1 NDD miRISC activity, with ALG-1 NDD miRISC sequestering functional miRNAs away from the ALG-2 miRISC. (B) Scatter plots of the total miRNA fold change (FC) versus NRF.score in the alg-1 NDD mutants. Dashed lines indicate two-fold differences between total miRNA FC and NRF.score. (C) Venn diagram of the miRNAs with lof NRF.score (<0.5) in the alg-1 NDD mutants. See also SI Appendix, Dataset S4 and Fig. S5.
Fig. 7.
Fig. 7.
The alg-1 NDD mutations have distinct impacts on gene target repressing modes. (A) Fold changes of mRNA abundance and TE of genes that are significantly up-regulated and contain target sites of miRNAs with lof NRF.score (<0.5). Dots indicate genes with significantly increased mRNA abundance (Cyan, |FC| > 1.5, p.adj < 0.1 by DEseq2), TE (Red, |FC| > 1.5, P < 0.1 by Student t test), or both TE and mRNA abundance (Magenta). (B) Summary of de-repression modes of genes that are translationally up-regulated and contain target sites for miRNAs with lof NRF.score. (C) Distribution of de-repression modes of genes in (B) with significantly up-regulated TE and/or significantly up-regulated mRNA abundance. (D) NRF.score of miRNAs that are down-regulated with statistical significance in both F180Δ and H751L mutants. (E) Fold changes of mRNA abundance and TE of genes that are translationally up-regulated and contain target sites of miRNAs in (D). (F) Distribution of the de-repression modes of the genes in E with significantly up-regulated TE and/or significantly up-regulated mRNA abundance. (G) Fold changes of mRNA abundance and TE of genes that contain target sites of miRNAs in (D) and were simultaneously perturbed in both alg-1(F180Δ) and alg-1(H751L) or both alg-1(G199S) and alg-1(H751L) mutants.
Fig. 8.
Fig. 8.
The alg-1 NDD mutations can perturb genes with human orthologs expressed in the brain and human orthologs related to NDD. (A) Venn diagram for genes that are translationally up-regulated in C.elegans and have human orthologs expressed in brain translatome (39). (B) MA plots for translationally perturbed genes with sysNDD curated human homologs (40). Solid and text labeled dots indicate genes that contain definitive sysNDD entity. Dot radius indicates the sysNDD entity counts. See also Dataset S5. (C and D) Hypergeometric tests for enrichment of stress-related genes (A) and unfolded protein response (UPR) (B) in translationally up-regulated genes in the alg-1 NDD mutants (41). (E) Summary of possible contributions of the alg-1/AGO1 NDD mutations to the pathogenesis of NDD.
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