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. 2004 Apr;165(1):31-40.
doi: 10.1083/jcb.200309008. Epub 2004 Apr 5.

Cytoplasmic Foci Are Sites of mRNA Decay in Human Cells

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Free PMC article

Cytoplasmic Foci Are Sites of mRNA Decay in Human Cells

Nicolas Cougot et al. J Cell Biol. .
Free PMC article

Abstract

Understanding gene expression control requires defining the molecular and cellular basis of mRNA turnover. We have previously shown that the human decapping factors hDcp2 and hDcp1a are concentrated in specific cytoplasmic structures. Here, we show that hCcr4, hDcp1b, hLsm, and rck/p54 proteins related to 5'-3' mRNA decay also localize to these structures, whereas DcpS, which is involved in cap nucleotide catabolism, is nuclear. Functional analysis using fluorescence resonance energy transfer revealed that hDcp1a and hDcp2 interact in vivo in these structures that were shown to differ from the previously described stress granules. Our data indicate that these new structures are dynamic, as they disappear when mRNA breakdown is abolished by treatment with inhibitors. Accumulation of poly(A)(+) RNA in these structures, after RNAi-mediated inactivation of the Xrn1 exonuclease, demonstrates that they represent active mRNA decay sites. The occurrence of 5'-3' mRNA decay in specific subcellular locations in human cells suggests that the cytoplasm of eukaryotic cells may be more organized than previously anticipated.

Figures

Figure 1.
Figure 1.
Distribution of various mRNA decay factors in human cells. Localization of GFP-hDcp1b (A), GFP-hLsm1 (B), GFP-hLsm3 (C), GFP-rck/p54 (D), GFP-hCcr4 (E), and GFP-DcpS (F). Left panels show GFP signal, middle panels show staining of the nucleus with propidium iodide, and right panels show overlays of the two signals. Different magnifications were used for the various panels to enhance visibility of important features. Some typical foci are indicated by arrowheads.
Figure 2.
Figure 2.
Colocalization of mRNA decay factors with hDcp1a. HEK293 cells were transiently transfected with GFP-hDcp1b (A), GFP-hLsm1 (B), GFP-hLsm3 (C), GFP- rck/p54 (D), or GFP-hCcr4 (E) and counterstained for hDcp1a. Left panels show GFP signals. Middle panels show immunostaining with rabbit anti-hDcp1a serum (1:1,000) and Fluorolink Cy5-labeled goat anti–rabbit IgG (1:1,000). Right panels show overlays of the two signals with arrowheads pointing to colocalizing proteins. Different magnifications were used for the various panels.
Figure 3.
Figure 3.
hDcp1a-containing bodies are not stress granules. Permeabilized HEK293 cells were incubated with rabbit anti-hDcp1a serum and anti–hTIA-1 mAbs (see Material and methods). (A) Untreated cells. (B) Cells treated with 0.5 mM sodium arsenite for 30 min. Left panels show the hDcp1a signal, middle panels show the hTIA-1 signal, and right panels show overlays of these two signals. Bars, 20 μm.
Figure 4.
Figure 4.
hDcp1a and hDcp2 interact in cytoplasmic foci. (A) Example of a FRET experiment where a fusion protein, CFP-YFP, was expressed in HEK293 cells as a positive control. A picture of the YFP signal was taken after photobleaching of a rectangular sector overlapping the cell (outlined in the CFP signal picture, bottom). A picture of the CFP signal was taken before (top right) and after (bottom) photobleaching of the YFP. Apparent FRET efficiency (18% for this experiment) was calculated as described in Materials and methods. (B) Example of a FRET experiment where CFP-hDcp1a and YFP-hDcp2 were expressed in HEK293 cells. Pictures were taken as described in A. Quantification of a foci in the bleached region shows an apparent FRET efficiency of 17% for this experiment (bottom left), whereas quantification of a foci out of the bleached region shows FRET efficiency of 4% (bottom right). (C) Histogram representing the average apparent FRET efficiency for the different constructions in different experiments. Error bars indicate the standard deviation from the independent measurements.
Figure 5.
Figure 5.
hDcp1a-containing bodies disappear after treatment with cycloheximide. HEK293 cells stably expressing hDcp1a were treated with 5 μg/ml cycloheximide for 5, 15, 30, and 120 min. Six fields randomly chosen were observed per time point, and number of foci per cell (covering ∼170 cells) was counted and reported on a histogram. (A) Distribution of hDpc1a at time 0. (B) Distribution of hDcp1a after 2 h of treatment. Bars, 20 μm. (C) Histogram representing a time course evolution of the average number of foci. Black bars represent untreated cells, gray bars represent treatment with DMSO, white bars represent treatment with cycloheximide, and the shaded bar represents cells at time 0 before any treatment.
Figure 6.
Figure 6.
hDcp1a-containing bodies disappear after treatment with actinomycin D. HEK293 cells stably expressing hDcp1a were treated with 5 μg/ml actinomycin D for 2, 4, 8, and 24 h. Six fields randomly chosen were observed per time point and number of foci per cell was counted and reported on a graph. (A) Distribution of hDpc1a at time 0. (B) Distribution of hDcp1a after 24 h of treatment. Bars, 10 μm. (C) Histogram representing time course evolution of the average number of foci. Black bars represent treatment with DMSO, white bars represent treatment with actinomycin D, and the shaded bar represents cells at time 0 before any treatment.
Figure 7.
Figure 7.
poly(A) + RNA accumulate in hDcp2-containing bodies after RNAi-mediated Xrn1 inactivation. (A) Northern blotting analysis of Xrn1 expression in control and Xrn1-inactivated cells. GAPDH mRNA was used as a loading control. Note that as the shRNA- expressing construct is stably integrated, reduced mRNA levels are expected to translate into corresponding reduced protein levels independently of the protein half-life except in the case of feed-back control. (B) poly(A)+ RNA detection by FISH in control cells (left) and Xrn1-inactivated RNAi cells (right). Arrowheads indicate sites of accumulation of poly(A)+ RNAs that may originate from incomplete deadenylation before decapping (Daugeron et al., 2001), overflow of the mRNA decay machinery resulting from Xrn1 inactivation, and/or degradation of NMD substrates. (C) Colocalization of GFP-hDcp2 and poly(A)+ mRNA accumulation sites. Left panels show GFP signal of GFP-hDcp2 fusion, middle panels show Cy5 signal corresponding to poly(A)+ RNAs, and right panels show overlay of the two signals, with arrowheads indicating colocalization. Bars, 4 μm.

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