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. 2006 Nov 28;103(48):18125-30.
doi: 10.1073/pnas.0608845103. Epub 2006 Nov 20.

Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules (V体育安卓版)

Anthony K L Leung  1 , J Mauro Calabrese, Phillip A Sharp
Affiliations

VSports最新版本 - Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules

Anthony K L Leung et al. Proc Natl Acad Sci U S A. .

Abstract

Argonaute proteins associate with microRNAs (miRNAs) that bind mRNAs through partial base-pairings to primarily repress translation in animals. A fraction of Argonaute proteins and miRNAs biochemically cosediment with polyribosomes, yet another fraction paradoxically accumulates in ribosome-free processing bodies (PBs) in the cytoplasm. In this report, we give a quantitative account of the Argonaute protein localization and dynamics in living cells in different cellular states. We find that the majority of Argonaute is distributed diffusely in the cytoplasm, and, when cells are subjected to stress, Argonaute proteins accumulate to newly assembled structures known as stress granules (SGs) in addition to PBs. Argonaute proteins displayed distinct kinetics at different structures: exchange faster at SGs and much slower at PBs. Further, miRNAs are required for the Argonaute protein localization to SGs but not PBs VSports手机版. These quantitative kinetic data provide insights into miRNA-mediated repression. .

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Conflict of interest statement

The authors declare no conflict of interest.

"V体育2025版" Figures

Fig. 1.
Fig. 1.
Ago2 localizes to the cytoplasm, PBs, and SGs upon inhibition of translation initiation. (a and b) Stably expressed EGFP-Ago2 localized to the cytoplasm and PBs (arrows) (a) and, upon addition of 1 μM hippuristanol (HIPP) for 30 min, also localized to SGs (arrowheads) (b). (c) The localization of EGFP-Ago2 is the same as the endogenous Ago2 in the cytoplasm and PBs. (d–f) Upon addition of 1 μM HIPP, EGFP-Ago2 colocalized with PB marker Dcp1a (d) and SG marker TIA1 (e), and the endogenous Ago2 also colocalized with SG marker TIA1 in parental HeLa cells (f). (Scale bars, 5 μm.)
Fig. 2.
Fig. 2.
Quantitative dynamics of Ago2 at PBs and SGs. (a) Time-lapse micrographs of stably expressed EGFP-Ago2 in a single live cell (complete movie is available in Movie 1). The first appearance of EGFP-Ago2 at SGs (arrowheads) occurred between 5 and 6 min after addition of 1 μM hippuristanol (HIPP). (b) Fluorescence recovery after photobleaching (FRAP) analyses of EGFP-Ago2 at single PBs (orange; n = 5) and SGs (purple; n = 3) and the intensities at respective structures relative to their initial intensities were compared over time. (c and d) 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. (c) The intensities of PAGFP-Dcp1a (orange; n = 3) and PAGFP-Ago2 (blue; n = 3) at the photoactivated PBs were compared over time with their respective initial intensities after photoactivation. (d) Representative images of PAGFP-Ago2 (Upper) and PAGFP-Dcp1a (Lower) at t = 00:00 and 12:54. The intensity of PAGFP-Dcp1a decreased over time at the photoactivated PBs (arrows), whereas the one of PAGFP-Ago2 remained relatively constant over time. Also, it was observed that the intensities of PAGFP-Dcp1a gradually increased over time at the neighboring PBs (broken arrows; see also Fig. 7e), suggesting continuous exchange of Dcp1a between PBs and the cytoplasm. (e) Addition of emetine reduced the accumulation of EGFP-Ago2 at SGs labeled by TIA1 (red) even in the presence of continual oxidative stress (250 μM arsenite for 90 min). In this experiment, 10 μg/ml emetine (Right) or DMSO (Left) were added 30 min after the arsenite treatment and left for 60 min further before fixing the cell for immunolabeling with SG marker TIA1 and PB marker Dcp1a (data not shown). (Scale bars, 5 μm.)
Fig. 3.
Fig. 3.
Short RNAs acting as miRNAs/siRNAs enriched at SGs. (a) HeLa cells electroporated with 0.83 μM quenched TAMRA siCXCR4 together with different luciferase constructs containing either no binding site for siCXCR4 (no target), one perfect binding site (siRNA target), or six bulged binding sites (miRNA target) were fixed after 56 h and immunolabeled with PB marker Dcp1a. siCXCR4 did not colocalize with PBs (Left, arrows) and there was no enrichment of intensity observed at PBs relative to the cytoplasm (i.e., relatively intensity = 1; Right, blue). As a control, a similarly quenched TAMRA-labeled DNA/RNA hybrid was used to measure the degree of nonspecific nucleic acid binding (red). Note that the siCXCR4 still targets endogenous genes even though the cotransfected Renilla luciferase has no CXCR4 binding site (no target). (b) Similarly, the localization of siCXCR4 was compared with PB marker Dcp1a (arrows) and SG marker TIA1 upon addition of 1 μM hippuristanol (HIPP) for 30 min. In this case, siCXCR4 colocalized 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 (Right; two-tailed t test, P ≪ 0.0001). (Scale bars, 5 μm.)
Fig. 4.
Fig. 4.
The localization of Ago2 at SGs, but not at PBs, depends on the presence of short RNAs. (a) Dicer+/+ (Upper) and Dicer−/− (Lower) cells transiently transfected with EGFP-Ago2 were fixed after 24 h and immunolabeled with PB marker Dcp1a. In both cases, EGFP-Ago2 colocalized with PBs (arrows). (b) Upon addition of 1 μM hippuristanol (HIPP), transiently expressed EGFP-Ago2 still colocalized with PB marker Dcp1a in both Dicer+/+ (Top, arrows) and Dicer−/− (Middle and Bottom) cells. However, EGFP-Ago2 no longer colocalized with SG marker TIA1 (arrowheads) in Dicer−/− cells. Cotransfection of 100 nM of miRNA let7a in the form of siRNA duplex resulted in the localization of EGFP-Ago2 at SGs in Dicer−/− cells (Bottom). (c) 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 b, 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.)
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