Quantifying Argonaute proteins in and out of GW/P-bodies: implications in microRNA activities

Abstract

MicroRNAs (miRNAs) are a class of ∼22nt non-coding RNAs that regulate the translational potential and stability of mRNAs. Though constituting only 1-4% of human genes, miRNAs are predicted to regulate more than 60% of all mRNAs. The action of miRNAs is mediated through their associations with Argonaute proteins and mRNA targets. Previous studies indicated that though the majority of Argonaute proteins is diffusely distributed in the cytoplasm, a small fraction is consistently observed to be concentrated in a cytoplasmic compartment called GW/P-bodies. In this chapter, we will provide a quantitative and dynamic view of the subcellular localization of miRNA function, followed by a discussion on the possible roles of PBs in miRNA silencing.

Figure 10.1

Schematics of (a) microRNA biogenesis and (b) Argonaute protein domains

Figure 10.2. Argonaute protein localization

(a) Stably expressed EGFP-Ago2 localized to the cytoplasm and PBs (arrows, left) and, upon addition of 1 μM hippuristanol (HIPP) for 30 min, also localized to SGs (arrowheads, right). (b) Time-lapse micrographs of stably expressed EGFP-Ago2 in a single live cell. The first appearance of EGFP-Ago2 at SGs (arrowheads) occurred between 5 and 6 min after addition of 1 μM hippuristanol (HIPP). Scale bars, 5 μm. (Reprinted, with permission, from Leung et al. (2006) PNAS 103:18125-30; © National Academy of Sciences, U.S.A.)

Figure 10.3. Localization of short RNAs

(a) Schematics of active siRNA strand detection. (b) Fluorescence emission profiles of unquenched and quenched TAMRA siRNAs against endogenous gene CXCR4 (siCXCR4). (c-d) The localization of siCXCR4 was compared with PB marker Dcp1a (arrows) and SG marker TIA-1 upon addition of 1 μM hippuristanol (HIPP) for 30 min. In this case, siCXCR4 co-localized with SGs (arrowheads), but not with PBs, when translation initiation was inhibited (Left), as shown by the significant enrichment of the intensities at SGs compared with the cytoplasm (two-tailed t test, P ≪ 0.0001). Scale bars, 5 μm. (Reprinted, with permission, from Leung et al. (2006) PNAS 103:18125-30; © National Academy of Sciences, U.S.A.)

Figure 10.4. Quantitative Dynamics of Argonaute protein

(a) FRAP analyses of EGFP-Ago2 at single PBs (n = 5) and SGs (n = 3) and the intensities at respective structures relative to their initial intensities were compared over time. (b) PAGFP-Dcp1a and PAGFP-Ago2 were photoactivated at single PB labeled by mRFP-Dcp1a for 1 s, and the photoactivated cells were imaged over the next 13 min. (Reprinted, with permission, from Leung et al. (2006) PNAS 103:18125-30; © National Academy of Sciences, U.S.A.)

Figure 10.5. The localization of Ago2 at SGs, but not at PBs, depends on the presence of short RNAs

(a) Transiently expressed EGFP-Ago2 still co-localized with PB marker Dcp1a in both Dicer^+/+ (Top, arrows) and Dicer^−/− (Middle and Bottom) cells. However, EGFP-Ago2 no longer co-localized with SG marker TIA-1 (arrowheads) in Dicer^−/− cells. Cotransfection of 100 nM of miRNA let-7a in the form of siRNA duplex resulted in the localization of EGFP-Ago2 at SGs in Dicer^−/− cells (Bottom). (b) The intensities of EGFP-Ago2 at SGs were compared with the cytoplasm in each case, and a histogram was plotted with the percentage of SGs as the y axis, for each relative intensity with an interval of 0.05 difference on the x axis. Correlated with the image data in (a), the intensities of EGFP-Ago2 at SGs relative to the cytoplasm were centered at ≈1.0 in Dicer^−/− cells, suggesting that there is no enrichment of EGFP-Ago2 at SGs in the absence of short RNAs. Scale bars, 5 μm. (Reprinted, with permission, from Leung et al. (2006) PNAS 103:18125-30; © National Academy of Sciences, U.S.A.)