Colocalization of microinjected miRNAs with PBs and cognate mRNA targets. Colocalization of microinjected miRNAs with PBs and cognate mRNA targets. (A)

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Colocalization of microinjected miRNAs with PBs and cognate mRNA targets. Colocalization of microinjected miRNAs with PBs and cognate mRNA targets. (A) miRNA–PB colocalization. Differential interference contrast (left) and pseudo‐coloured images depicting the colocalization (overlay, right) of ectopically expressed EGFP‐tagged Dcp1a, a component of PBs (green), and microinjected let‐7a‐1–Cy5 miRNA (red). Wide‐field epi‐illumination was used to visualize these cells. Arrows show representative foci containing both Dcp1a and miRNA. Scale bar, 10 μm. (B) Schematic of an EGFP‐labelled mRNA (top) based on the MS2–MS2 coat protein (MCP) mRNA‐labelling system [[16]]. (C) miRNA–mRNA colocalization. Left, composite image of GFP (green) and Cy5 (red) channels from a representative cell expressing the mutant–mRNA–EGFP and mutant let‐7a‐1–Cy5 miRNA. Yellow particles indicate colocalization. Scale bar, 5 μm. Centre, colocalization map representing independent miRNA (red) and mRNA (GFP) particles, and either perfectly colocalized (yellow) or adjacent miRNA–mRNA localizations with an interparticle centre–centre distance less than the lateral resolution limit of the image (cyan and magenta, supplementary Methods online). Right, zoomed view of the region in the preceding images marked by a white box. Yellow circles indicate colocalized spots; scale bar, 1 μm. (D) Fraction of specific or nonspecific miRNA colocalizing with mRNA–EGFP. Cells were injected with the MS2/MCP plasmids and either mutant (specific; circle) or wild‐type (nonspecific; square) let‐7a‐1. To test for the contribution of background fluorescence, control cells were injected with mutant let‐7a‐1 and MCP plasmid without the MS2–mRNA expression plasmid (specific; triangle). Data points within each group and their associated means (dashed lines) are shown (n=2 cells for each group). EGFP, enhanced green fluorescent protein; miRNA, microRNA; PB, processing body. Sethuramasundaram Pitchiaya et al. EMBO Rep. 2012;13:709-715 © as stated in the article, figure or figure legend