Figure 4: LINE-1 RNA in P-bodies or stress granules is preferentially targeted for autophagic degradation by NDP52 and P62, respectively. | Nature Communications

Figure 4: LINE-1 RNA in P-bodies or stress granules is preferentially targeted for autophagic degradation by NDP52 and P62, respectively.

From: Autophagy supports genomic stability by degrading retrotransposon RNA

Figure 4

(a,b) Fluorescent microscopy of (a) NDP52 or (b) P62 in cells expressing LINE-1-MS2 and MS2-GFP. (c,d) Fluorescent microscopy of endogenous LINE-1 RNA and (c) NDP52 or (d) P62. (e) Four-colour confocal microscopy of NDP52, P62, GFP-DCP1A and mCherry-TIA-1. (f) Percent of foci of NDP52, P62 or neither co-localizing with GFP-DCP1A or mCherry-TIA-1. *P=0.007 GFP-DCP1A, P=0.00005 mCherry-TIA-1, analysis of variance (ANOVA). (g) Percent of foci of NDP52, P62 or both co-localizing with GFP-DCP1A or mCherry-TIA-1, n=674 stress granules, n=53 P-bodies. *P=0.043 GFP-DCP1A, P=0.028 mCherry-TIA-1, ANOVA. (h) Venn diagram of the percent of P-bodies (GFP-DCP1A) or stress granules (mCherry-TIA-1) co-localizing with NDP52, P62, both or neither. (i) Relative number of P-bodies per cell (detected with 18033 serum-recognizing GW182) treated with indicated siRNAs. n=3, *P=0.0002 NDP52, P=0.004 ATG5. (j) Relative number of stress granules (detected with antibody recognizing TIAR) 1 h after arsenite treatment and after 30 min recovery from 1 h treatment with arsenite. n=3, *P=4x10−6 p62, P=0.019 NDP52, P=2 × 10−5 ATG5, ANOVA. (k) Percent of cells expressing GFP when co-transfected with LINE-1-RP retrotransposition GFP reporter and either of two independent siRNA (black and grey bars) targeting NDP52, P62 or control. Results are normalized to a retrotransposition-incompetent control (n=3, *P=0.017, P=0.002 NDP52; P=0.001, P=0.013 p62, ANOVA). (l) Relative number of G418-resistant colonies in cells transfected with siRNA-targeting NDP52, P62 or control, Alu-retrotransposition reporter and retrotransposition-enhancer LINE-1 ORF2p (n=3, *P=0.012, P=0.011 NDP52; P=0.008, P=0.011 p62, ANOVA). All error bars represent s.e. of the mean. All experiments were performed with a minimum of three biological replicates.

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