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AGO2 silences mobile transposons in the nucleus of quiescent cells

Nature Structural & Molecular Biology (2023)Cite this article

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Abstract

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway, with essential roles in development and disease. Yet little is known about its regulation in vivo. Here we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K–AKT–mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells in which mTOR signaling is low, AGO2 accumulates in the nucleus, where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

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Fig. 1: AGO2 is a nuclear protein in resting mouse splenocytes.
Fig. 2: Regulation of AGO2 subcellular localization.
Fig. 3: AGO2 localization is regulated by the Pi3K–AKT–mTOR pathway.
Fig. 4: Nuclear AGO2 binds young retrotransposable elements.
Fig. 5: AGO2 associates with TE transcripts in quiescent cells.
Fig. 6: AGO2 associates with TE-derived small RNAs in quiescent cells.
Fig. 7: AGO2 represses LINE-1 elements in quiescent cells via its catalytic domain.

Data availability

FASTQ and processed files from ChIP–seq and RNA sequencing datasets produced in this study can be found at the Gene Expression Omnibus (GEO) (GSE203049).

ChIP–seq data for H3K9me3 in resting splenic B cells shown in Figure 4 can be downloaded from the GEO (GSE82144). Source data are provided with this paper.

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Acknowledgements

We thank all members of the Vidigal and Batista labs for discussions and comments on this work. We also thank P. P. Rocha, S. Chakraborty, and J. Thompson for help and advice on ChIP–seq experiments. We thank the NCI’s Laboratory Animal Sciences Program, in particular D. Gallardo and M. Figueroa, for expert mouse care and help maintaining the mouse colonies. We also thank the NCI’s molecular histopathology core, especially T. Morgan, J. Mata, and B. Karim. This work utilized the computational resources of the NIH HPC Biowulf cluster (hpc.nih.gov). R.L.C. is supported by the NIH PRAT fellowship FI2GM142571-01. This work was supported by the Intramural Research Program of the National Institutes of Health through the Center for Cancer Research, National Cancer Institute, project 1ZIABC011810-02 (J.A.V.), and was partially funded by contract number HHSN261201500003I (R.C., P.A.).

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  1. These authors contributed equally: Laura Sala, Manish Kumar, Mahendra Prajapat.

Authors and Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, The National Institutes of Health, Bethesda, MD, USA

    Laura Sala, Manish Kumar, Mahendra Prajapat, Srividya Chandrasekhar & Joana A. Vidigal

  2. The Eunice Kennedy Shriver National Institute of Child Health and Human Development, The National Institutes of Health, Bethesda, MD, USA

    Rachel L. Cosby & Todd S. Macfarlan

  3. The National Institute for General Medical Sciences, The National Institutes of Health, Bethesda, MD, USA

    Rachel L. Cosby

  4. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Gaspare La Rocca

  5. Laboratory Animal Sciences Program, Frederick National Lab for Cancer Research, The National Institutes of Health, Frederick, MD, USA

    Parirokh Awasthi & Raj Chari

  6. CCR Confocal Microscopy Core Facility, National Cancer Institute, The National Institutes of Health, Bethesda, MD, USA

    Michael Kruhlak

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  1. Laura Sala
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Contributions

L.S. and J.A.V. conceived the project with input from G.L.R. L.S., M. Kumar, M.P., and J.A.V. performed experiments and analysis. S.C., R.C., M. Kruhlak, and P.A. helped with experiments. J.A.V. supervised the project. R.L.C. and T.S.M. provided expertise in the analysis over TE elements. L.S. and J.A.V. wrote the manuscript with input from all authors.

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Correspondence to Joana A. Vidigal.

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Extended data

Extended Data Fig. 1 Ago2HA/HA mice are phenotypically normal and have intact Ago2 functions.

(a) Targeting strategy. An 2xHA tag and short linker sequence were introduced directly downstream of start codon maintaining 5′UTR and promoter sequences intact. Position of the first exon (1) and length of homology arms are shown. (b) Western blot to MEFs obtained from heterozygous intercrosses. (c) Luciferase assay in MEFs showing intact repression of miRNA (orange) and AGO2 cleavage (red) reporters. Values represent mean of two biological replicates. Error bars represent standard deviation. p-values were calculated using a one-sided t-test. (d) Expected and observed numbers of genotypes obtained from heterozygous intercrosses at weaning. p-value was calculated with a Chi-square test. (e) Weigh of littermate animals at weaning (males: 14 wild-type, 25 heterozygous, 12 homozygous; females: 11 wild-type, 30 heterozygous, 9 homozygous). Data are presented as mean values +/− SD. p-values were calculated using a one-sided t-test. (f) Hematoxylin & Eosin-stained sections of tissues isolated from Ago2HA/HA animals and Ago2+/+ controls showing intact tissue morphology in homozygous animals.

Source data

Extended Data Fig. 2 AGO2 localization in quiescent versus proliferating cells.

(a) Ex-vivo splenocyte activation leads to a significant increase in cell size. (b) Splenocyte activation has minimal impact in relative sizes of nuclear and cytoplasmic compartments. (c) Percentage of total HA signal that is localized to the nucleus in activated B and T splenocytes. (d) Percentage of total HA signal that is localized to the nucleus in primary MEFs grown in complete media (10% serum; Fed), serum starved for 8 days (0.1% serum; Starved, Str), and refed for 24 h following an 8-day serum starvation (Refed, Ref). For all boxplots, each dot represents a cell. Boxplots show minimum, maximum, median, first, and third quartiles. Median value (x) and number (n) of cells analyzed per condition are shown. Values are based on 93 resting and 154 activated B cells or 141 resting and 69 activated T cells collected from two independent experiments. P values were calculated with a two-sided Wilcoxon test.

Source data

Extended Data Fig. 3 AGO2 is regulated by the Pi3K-AKT-mTOR pathway at least in part through levels of TNRC6.

(a) Chemical inhibition of Pi3K-AKT-mTOR and MEK-ERK pathways in immortalized MEFs. A schematic representation of the Pi3K pathway and the drugs used for its inhibition is shown on the left (Pi3Ki, LY294002; AKTi, MK2206; mTORi, Torin1). (b) Top, schematic representation of the MEK-ERK mitogenic pathway and the drug used to inhibit is (MEKi, PD032590). Bottom, western blot showing activity of the pathway in resting (Res) and activated (Act) splenocytes. (c) Western blot showing AGO2 and TNRC6 levels in primary MEFs cultured in complete media (Fed), serum starved (Str), or starved and refeed for 24 h (Ref). (d) TNRC6 levels but not AGO2 levels are downregulated by Pi3K pathway inhibitors. (e) qPCR to Tnrc6 family members following the delivery of indicated siRNAs to immortalized MEFs. Data are presented as mean values of three independent experiments +/− SD. p-values were calculated using a two-sided Wilcoxon test. (f) Western blot to TNRC6 following downregulation of all three isoforms.

Source data

Extended Data Fig. 4 AGO2HA associates with chromatin in an RNA-dependent manner.

(a) Western blot showing undetectable expression of AGO1 and AGO3 in primary mouse B cells. Lysates from two biological independent mouse embryonic fibroblast lines (MEF1 and MEF2) were used as control. Res., resting cells; Act., activated cells. (b) Schematic representation of experimental procedure. RNase treatment was performed on nuclear extracts before separating nuclear soluble (NS) and chromatin enriched (Chr) fractions. As control, samples from wild-type or tagged animals were processed in parallel but omitting the RNA endonucleases (mock). (c) Western blot to sub-cellular fractions following RNase treatment of nuclear extracts shown in (B).

Source data

Extended Data Fig. 5 Enrichment of AGO2HA peaks over TE elements.

(a) MACS-called peaks overlap almost exclusively with repetitive elements. (b) Significance values for all repetitive elements that are significantly enriched in high-confidence AGO2 peaks. (c) Mean expected overlaps (calculated from 1000 random shuffles) compared to the observed overlaps over significantly enriched LINE elements. (d) Mean expected overlaps (calculated from 1000 random shuffles) compared to the observed overlaps over significantly enriched ERV elements.

Source data

Extended Data Fig. 6 AGO2HA is enriched at young LINE and ERV retrotransposons.

(a) Example genome browser view showing enrichment of AGO2HA over the consensus sequence of young LINE-1 elements (blue) or young ERV elements (green). For each bin, enrichment was calculated as the log2 value of the ratio between the reads per million (RPM) of AGO2HA samples (HARPM) and those of the wild-type samples (WTRPM). As a result, regions where AGO2HA is enriched are represented by positive values, and those where AGO2HA is depleted are represented by negative values. (b) Example genome browser view over a young L1 element for the indicated ChIP-seq data. Individual biological replicates are shown in light blue (Ago2HA/HA samples) or light grey (Ago2+/+ samples). Tracks with replicate data merged are show in dark blue or dark grey. Notice the enrichment of ChIP-seq reads in Ago2HA/HA samples over the LINE-1 element compared to control Ago2+/+ samples when all reads (uniquely mapping as well as multimapping) are considered. In contrast, because of its repetitive nature, reads mapping uniquely to this element are mostly absent. Notice also how paired-end sequencing (PE seq) which increases the length of the reads compared to single-read (SR) allows the identification of a peak for uniquely mapping reads at the border between the L1 element and the uniquely mappable region of the genome in the Ago2HA/HA but not the Ago2+/+ ChIP confirming that enrichment is not an artefact of multimapping reads. A detailed view of this peak as well as the underlying reads is show in (c).

Extended Data Fig. 7 ChIP-seq analysis to AGO2HA in quiescent B cells.

(a) Correlation of normalized read counts across biological replicates mapping to transposable elements (TE) following ChIP-seq with an antibody against HA in Ago2+/+ (left) and Ago2HA/HA (right). Each dot represents a distinct TE. Repeats enriched in Ago2HA/HA compared to Ago2+/+ ChIP-seq are labeled in red. (b) As in (A) but comparing Ago2HA/HA and Ago2+/+ normalized read counts for replicate 1 (left) and 2 (right). (c) Cumulative Distribution Fraction plot for log2(TPM + 1) expression values of repeats with (red) or without (grey) AGO2HA enrichment in ChIP-seq data. TPM, transcript per million. p-value was calculated with a two-sided Kolmogorov–Smirnov test.

Extended Data Fig. 8 Small RNAs associated with AGO2 in resting and activated cells.

(a) Representative western blot of fractions obtained following AGO2 pulldowns. Sup., supernatant; Inp., input; IP, immunoprecipitation. 1% of each fraction was loaded on the blot. Inputs from wild-type controls are shown as a control for the specificity of the antibody. (b) Composition of small RNA reads that map to annotated ncRNA loci in input (left) and IP samples (right) according to length distribution. Reads were normalized to total number of mapped reads. (c) Enrichment of miRNAs in AGO2 pulldowns in activated (Act.) and resting (Res.) primary B cells. (d) Number of reads mapping to miRNAs or TEs following pulldowns of AGO2 in resting B cells. The y-axis represents reads per million (RPM) of 20-24 nucleotide long RNAs. Data shown in C-D represents the average between two biological replicates.

Source data

Extended Data Fig. 9 Loss of Ago2 catalytic competence does not result in overt changes in H3K9me3.

(a) Western blot to AGO2 following CRE-mediated locus recombination. (b) Left, representation of the outcome of the recombination experiment. Right, relative expression levels of IAP and MERVL following conditional loss of AGO2 in resting B cells. Data are presented as mean of biological duplicates +/− SD. p values were calculated with a two-sided t-test. (c) Profile plots showing normalized read counts for H3K9me3 ChIP-seq over two example young L1 repeats following CRE-mediated recombination of Ago2flx/+ and Ago2flx/ADH in resting B cells and Ago2+/+ controls. (d) H3K9me3 ChIP signal depth over consensus repeat sequences. (e) Enrichment for H3K9me3 (calculated as log2 of the ratio between ChIP and input) for repeats of the L1MdA (top), L1MdTf, and L1MdGf (bottom) young L1 families following CRE-mediated recombination of Ago2flx/+ and Ago2flx/ADH in resting B cells and Ago2+/+ controls. Boxplots show minimum, maximum, median, first, and third quartiles from two biological replicates whose individual values are represented by a dot.

Source data

Extended Data Fig. 10 Overlap of AGO2HA and histone post-translational modifications.

Representative SIM super-resolution optical mid-sections of co-immunofluorescence for HA (green) and the indicated histone marks (red). Scale bar: 1 µm.

Supplementary information

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Supplementary Figs. 1–3, Table 1, Note 1 and Methods.

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Source data

Source Data Figs. 1 and 2 and Extended Data Fig. 2

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Sala, L., Kumar, M., Prajapat, M. et al. AGO2 silences mobile transposons in the nucleus of quiescent cells. Nat Struct Mol Biol (2023). https://doi.org/10.1038/s41594-023-01151-z

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