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Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA

Nature Structural & Molecular Biology volume 21, pages 552–559 (2014)Cite this article

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Abstract

Dicer is a central enzymatic player in RNA-interference pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, and its nuclear levels are tightly regulated. Dicer interacts with RNA polymerase II (Pol II) at actively transcribed gene loci. Loss of Dicer causes the appearance of endogenous double-stranded RNA (dsRNA), which in turn leads to induction of the interferon-response pathway and consequent cell death. Our results suggest that Pol II–associated Dicer restricts endogenous dsRNA formation from overlapping noncoding-RNA transcription units. Failure to do so has catastrophic effects on cell function.

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Figure 1: Dicer localizes in both the nucleus and cytoplasm of HEK293 cells.
Figure 2: Dicer interaction with Pol II is dsRNA dependent, and Dicer-chromatin association is transcription dependent.
Figure 3: Dicer affects levels of nascent transcripts and TGS.
Figure 4: Loss of Dicer leads to accumulation of long dsRNA.
Figure 5: Loss of Dicer leads to the accumulation of dsRNA that colocalizes with chromatin-associated Dicer loci.
Figure 6: Loss of Dicer leads to a reduction of siRNAs that colocalize with chromatin-associated Dicer sites and dsRNA sites.
Figure 7: Loss of Dicer triggers the interferon response.
Figure 8: Model of nuclear Dicer function.

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Acknowledgements

We are grateful to E. Johnson for help with transmission electron microscopy and scanning electron microscopy experiments. This work was supported by grants from the Wellcome Trust (091805/Z/10/Z to N.J.P.) and E.P. Abraham Trust (to N.J.P.) and by a Medical Research Council Career Development Award (MR/K006606/1 to M.G.) and a L'Oreal-UNESCO Woman in Science UK and Ireland award (to M.G.).

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Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Eleanor White, Margarita Schlackow, Kinga Kamieniarz-Gdula, Nick J Proudfoot & Monika Gullerova

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Contributions

E.W. and M.G. performed all the experimental analyses. M.S. and K.K.-G. performed bioinformatics analyses of the ChIP-seq and RNA-seq data. E.W., M.G. and N.J.P. designed the experiments and wrote the manuscript.

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Correspondence to Nick J Proudfoot or Monika Gullerova.

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Integrated supplementary information

Supplementary Figure 1 Dicer localizes in both nucleus and cytoplasm of HEK293 cells.

a) Western blot analysis of protein extracts from HEK293 cells with inducible integrated shRNA transgene. Induction by doxycycline leads to Dicer-specific shRNA production and consequent Dicer knockdown. Antibodies specific to Dicer and γ-actin were employed. b) Western blot analysis of Dicer in nuclear and cytoplasmic extracts from HEK293 cells transfected with episomal GFP-Dicer or RFP-Dicer expressing plasmids. Grp75 and γ-actin were used as controls. c) Expression of GFP-Dicer transgene (green) in normal and Dicer knockout murine embryonic stem cells as in Figure 1b. d) Control FRAP experiment shows Dicer-GFP (green) recovery in nucleus of HEK293 cells (lacking GFP signal) after bleaching. This provides a background control to FRAP analysis of GFP-Dicer expression in the nucleus of Dicer knockdown cells. Here, only background signals can be detected. The cell is shown in the left hand panel before bleaching. In the middle panel, the red circle corresponds to bleached area in nucleus. Fluorescent recovery was measured in the green circle. Fluorescent background values were measured in the blue circle. Fluorescent recovery was measured in 20 frames, bleaching was initiated at frame 5. Right-hand panel shows the cell after recovery. Absolute levels of fluorescence and relative levels of recovery (background fluorescence subtracted from fluorescence levels in measured region) are shown in the left and right panels, respectively. All experiments described in Supplementary Fig. 1a-c, were replicated three times.

Supplementary Figure 2 Genomic annotation of Dicer ChIP-seq peaks.

a) Table showing the top 12 peaks of Dicer binding loci according to the ChIP sequencing data. Nearest gene identity is shown. b) The 4 additional loci chosen for further analysis, showing a range of Dicer binding c) Metagene analysis of top 118 Dicer binding sites (ChIP-seq peaks) showing their location distribution around TSS and PAS of coding genes in the human genome. Data were grouped into 250 nt bins. Each bin-midpoint was plotted on the graph. d) Table summarising the enrichment of repetitive DNA sequence elements in the Dicer ChIP-seq data. The RepeatMasker hg19 table was used to extract genomic repetitive elements. Repetitive element overlapping a Dicer peak with 5 nt or more was included in the analysis. Each category of repetitive elements as presented, was analysed separately. p-values are based on a background distribution of sampling 118 random genomic sequences of the same length as Dicer peaks. Sampling was performed more than 1000 times. e) H3 control ChIP experiment as in Figure 2e. f) Top panel: Diagram of the MTRNR2L6. The start site of transcription (arrow) and the position of the PCR amplicons (bold) are indicated. Bottom panels: Pol II, Dicer and H3 ChIP experiments as in Figure 2e.

Supplementary Figure 3 Dicer and Pol II binding affect each other at selected loci.

a) qRT-PCR as in Fig 3d and 3e. Probes were specific to GAPDH and b) 28S rDNA.

Supplementary Figure 4 Loss of Drosha has no effect on the accumulation of long dsRNA.

a) IF as in Fig. 1a using anti-Drosha and J2 antibodies on normal and Drosha knockdown HEK293 cells. b) Levels of pri-mRNA15a transcripts as determined by qRT-PCR analysis using a specific primer for reverse transcription. RNA was isolated from normal HEK293 cells and cells treated with an shRNA against Drosha for 10 days. Signals are based on average values ± s.d. from three independent biological experiments and are normalised to the levels of transcripts in normal cells, set as 1.

Supplementary Figure 5 Loss of Dicer leads to accumulation of long dsRNA.

IF as in Fig. 1a using anti-Dicer and J2 antibodies on Dicer knockout murine embryonic stem cells.

Supplementary Figure 6 Loss of Dicer leads to accumulation of dsRNA.

a) Histogram of peak summit ratio of dsRNA in Dicer knockdown cells to dsRNA in normal cells. Any dsRNA signal >10 in both uninduced and induced cells was considered. Value higher than 1 indicates accumulation of dsRNA and value lower than 1 indicates a decrease in dsRNA levels in Dicer knockdown cells. The black line marks the value of 1. Dark blue bins indicate the number of times a ratio falls in value span marked on the horizontal axis. Light blue bins represent a cumulative number for the values under 1 or over 1 separately. Centromeric repeat regions were excluded from analysis. b) Diagram of the change level in dsRNA (according to peak summit value) accumulation in normal and Dicer knockdown cells. Any dsRNA signal >10 in both uninduced and induced cells was considered. Plotted levels from normal cells are subtracted Dicer knockdown cell levels. Value less than 0 (marked by a black vertical line) indicates the absolute value of the decrease in dsRNA, while a value greater than 0 indicates an increase. Each blue point depicts one locus of Dicer-dsRNA co-localization. The loci were ordered according to the difference value in ascending order. One point is too large to be depicted in the plot, it has value 2432530. Note the scale of the graph is (x104). Centromeric repeat regions were excluded from analysis. c) Box Plot of change in dsRNA levels (according to peak summit value) between Dicer knockdown and normal cells. Any dsRNA signal >10 in both uninduced and induced cells was considered. The red mark indicated the median, at a value of 855. The lower quartile q1 (25th percentile) is at -19 and the upper quartile q3 (75th percentile) at 3992 (blue box). The shift in the positive direction of the box shows a general increase in dsRNA at Dicer loci. 20 outlier values were excluded from the plot. Centromeric repeat regions were excluded from analysis. d) Tabular summary of enrichment of repetitive elements in dsRNA-seq data. The RepeatMasker hg19 table was used to extract genomic repetitive elements. Repetitive element overlapping a dsRNA peak with 5 nt or more was included in the analysis. Each category of repetitive elements as presented, was analysed separately. p-values are based on a background distribution of sampling 118 random genomic sequences of the same length as Dicer peaks. Sampling was performed more than 1000 times. e) Metagene analysis of top dsRNA peaks (see Online Methods) showing location distribution around TSS and PAS of coding genes in the human genome. Data were grouped into 250 nt bins. Each bin-midpoint was plotted on graph.

Supplementary Figure 7 Loss of Dicer triggers the interferon response.

a) Quantitation of Western blot analysis of cell extracts isolated from normal and Dicer knockdown HEK293 cells, growing in tissue culture for 1 or 2 weeks using antibodies specific to Dicer, TLR3, PKR1 and γ-actin (see Figure 7a). Signals were measured by image-Quant software, are expressed as a % of input and are expressed as an average of three independent experiments, normalised to normal HEK293 cell levels, set as 1. * indicates statistical significance (p < 0.05), based on unpaired, two-tailed distribution Student's t-test. b) IF as in Figure 4c using anti-PKR antibody (green).

Supplementary Figure 8 Loss of Dicer triggers the interferon response.

a) Morphological changes of normal and Dicer knockdown HEK293 cells using Electron Microscopy (SEM) at 3000-fold magnification. b) TEM analysis of nuclear structure in normal and Dicer knockdown HEK293. c) TEM as in middle panel focusing on mitochondria. Representative images from 20 cells are presented.

Supplementary Figure 9 Uncropped blot images.

a) Relating to Figure 2. b) Relating to Figure 4. c) Relating to Figure 6. d) Relating to Figure 7.

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Supplementary Figures 1–9 and Supplementary Table 1 (PDF 6583 kb)

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White, E., Schlackow, M., Kamieniarz-Gdula, K. et al. Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA. Nat Struct Mol Biol 21, 552–559 (2014). https://doi.org/10.1038/nsmb.2827

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