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. 2007 Jun;27(11):3970-81.
doi: 10.1128/MCB.00128-07. Epub 2007 Apr 2.

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing

Ana Eulalio  1 , Isabelle Behm-Ansmant, Daniel Schweizer, Elisa Izaurralde
Affiliations

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing

Ana Eulalio et al. Mol Cell Biol. 2007 Jun.

Abstract

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.

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Figures

FIG. 1.
FIG. 1.
Conservation of P-body components. (A and B) S2 cells were stained with affinity-purified anti-Tral or anti-Ge-1 antibodies. The antibody staining is specific, as it was strongly reduced in cells in which the corresponding proteins were depleted (knockdown). (B) The efficiency of Tral or Ge-1 depletions was assessed by Western blotting. (C) Confocal fluorescent micrographs of S2 cells expressing GFP fusions of the proteins indicated on the left (green channel). Cells were stained with affinity-purified anti-Tral antibodies (red channel). Scale bar, 5 μm.
FIG. 2.
FIG. 2.
Nontranslating mRNAs and overexpression of translational repressors enhance P-body formation. (A) Confocal fluorescent micrographs of S2 cells expressing GFP fusions of Staufen (Stau) or Smaug (green channel). Cells were stained with affinity-purified anti-Tral antibodies (red channel). Scale bar, 5 μm. (B) S2 cells were treated with RNase A (15 min, 100 μg/ml), cycloheximide (1 h, 10 μg/ml), or puromycin (1 h, 100 μg/ml) and stained with affinity-purified anti-Tral or anti-Ge-1 antibodies. (C) S2 cells were transfected with plasmids expressing F-Luc and R-Luc. Eight hours after transfection, cells were split into three pools. One pool was treated with puromycin, and one was treated with cycloheximide. Luciferase activity was measured 1 h after the addition of the drugs.
FIG. 3.
FIG. 3.
P-body formation requires mRNAs undergoing decapping. S2 cells from which the indicated proteins were depleted were stained with affinity-purified anti-Tral or anti-Ge-1 antibodies. Depletion of NOT1, LSm1, Me31B, HPat, or Ge-1 results in P-body loss, whereas depletion of DCP2 or XRN1 leads to an increase in P-body size. EDC3 or Tral depletions have no detectable effect on P-body integrity. Numbers indicate the fraction of cells exhibiting a staining identical to that shown in the representative panel in three independent knockdowns performed per protein (at least 100 cells were counted per knockdown). Scale bar, 5 μm.
FIG. 4.
FIG. 4.
P-body integrity depends on functional silencing pathways. S2 cells from which proteins involved in different steps of RNAi or the miRNA pathway were depleted were stained with affinity-purified anti-Tral or ant-Ge-1 antibodies. The fraction of cells exhibiting a staining identical to that shown in the representative panel was determined by scoring at least 100 cells in each of the three independent knockdowns performed per protein. Scale bar, 5 μm.
FIG. 5.
FIG. 5.
mRNAs that enter silencing pathways elicit P-body formation. (A) Drosha-depleted cells were transfected with SMG6 siRNA, F-Luc siRNA, or F-Luc siRNA together with a plasmid expressing F-Luc mRNA. A plasmid expressing GFP was cotransfected in order to visualize transfected cells. (B) AGO2-depleted cells were transfected with SMG6 siRNA together with a plasmid expressing GFP in order to visualize transfected cells. (C) S2 cells from which the proteins indicated on the left were depleted were treated with long dsRNAs targeting GFP or the Drosophila CG4415 mRNA. In all panels, cells were stained with affinity-purified anti-Tral antibodies. Numbers indicate the fraction of cells exhibiting a staining identical to that shown in the representative panel as described in the legends to Fig. 3 and 4. Scale bar, 5 μm.
FIG. 6.
FIG. 6.
Releasing mRNAs from polysomes is not sufficient for P-body formation. (A) AGO2-depleted cells were transfected with plasmids encoding pri-miR-12, pri-miR-12 together with F-Luc-CG10011 reporter, or the F-Luc-CG10011 reporter alone. A plasmid expressing GFP was cotransfected in order to visualize transfected cells. Cells were stained with affinity-purified anti-Tral antibodies. (B and C) AGO1-, LSm1-, or LSm3-depleted cells were transfected with plasmids encoding pri-miR-12 together with F-Luc-CG10011 reporter. A plasmid expressing GFP was cotransfected in order to visualize transfected cells. Cells were stained with affinity-purified anti-Tral antibodies. (D) S2 cells from which the proteins indicated on the left were depleted were treated with puromycin and stained with affinity-purified anti-Tral antibodies. Numbers indicate the fraction of cells exhibiting a staining identical to that shown in the representative panel as described in the legends to Fig. 3 and 4. Scale bar, 5 μm.
FIG. 7.
FIG. 7.
Silencing by miRNAs or dsRNAs does not require microscopic P bodies. (A) The indicated proteins were depleted from S2 cells (kd, knockdown). Depleted cells were cotransfected with plasmids expressing miRNA reporters (Nerfin or CG10011), plasmids expressing miR-9b or miR-12 or the corresponding empty vector. R-Luc served as a transfection control. F-Luc activity and the corresponding mRNA levels were measured and normalized to those of the Renilla control. Normalized F-Luc activities and mRNA levels in cells transfected with the empty vector were set to 100% for each knockdown (not shown). Error bars represent standard deviations from three independent experiments. (B) S2 cells expressing adh mRNA were treated with a dsRNA targeting a central region of the adh open reading frame or with GFP dsRNA as a control. The levels of adh mRNA were quantitated and normalized to those of a CAT mRNA transfection control. These values were set to 100 in cells treated with GFP dsRNA. The left panel shows results for S2 cells expressing adh mRNA that were treated with adh or GFP dsRNAs. These cells were then treated with dsRNAs targeting the proteins indicated. The levels of adh mRNA were quantitated and normalized to those of a transfection control (CAT mRNA). For each knockdown, the normalized values obtained for the cells in the presence of the adh dsRNA were divided by those obtained for cells treated with GFP dsRNA to compensate for unspecific effects of the depletions. Error bars represent standard deviations from three independent experiments. (C) Depletions shown in panel B resulted in P-body dispersion as shown by fluorescent staining of cells with anti-Tral antibodies. Numbers indicate the fraction of cells exhibiting a staining identical to that shown in the representative panel in the three independent knockdowns performed per protein (at least 100 cells were counted per knockdown). Scale bar, 5 μm.
FIG. 8.
FIG. 8.
NMD and mRNA decay do not require macroscopic P bodies. (A) The levels of endogenous ODA mRNA were analyzed by Northern blotting of cells from which the indicated proteins were depleted (knockdown [kd]). For each knockdown, ODA mRNA levels were normalized to the levels of rp49 mRNA. These ratios were set to 1 in control cells. Error bars represent standard deviations from three independent experiments. (B) S2 cells from which the indicated proteins were depleted (Kd) were transfected with the F-Luc-5BoxB reporter, a plasmid expressing R-Luc, and vectors expressing the λN peptide or λN-GW182. F-Luc activity and the corresponding mRNA levels were measured and normalized to those of the Renilla control. Normalized F-Luc activities and mRNA levels in cells transfected with the λN peptide alone were set to 100% for each knockdown (not shown). Error bars represent standard deviations from three independent experiments. We confirmed that the depletions shown in panels A and B resulted in P-body dispersion by fluorescent staining of cells with anti-Tral antibodies (see Fig. 3; data not shown).
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References

    1. Albrecht, M., and T. Lengauer. 2004. Novel Sm-like proteins with long C-terminal tails and associated methyltransferases. FEBS Lett. 569:18-26. - PubMed
    1. Anantharaman, V., and L. Aravind. 2004. Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability. BMC Genomics 5:45. - PMC - PubMed
    1. Anderson, P., and N. Kedersha. 2006. RNA granules. J. Cell Biol. 172:803-808. - PMC - PubMed
    1. Andrei, M. A., D. Ingelfinger, R. Heintzmann, T. Achsel, R. Rivera-Pomar, and R. Luhrmann. 2005. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 11:717-727. - PMC - PubMed
    1. Barbee, S. A., P. S. Estes, A. M. Cziko, J. Hillebrand, R. A. Luedeman, J. M. Coller, N. Johnson, I. C. Howlett, C. Geng, R. Ueda, A. H. Brand, S. F. Newbury, J. E. Wilhelm, R. B. Levine, A. Nakamura, R. Parker, and M. Ramaswami. 2006. Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies. Neuron 52:997-1009. - PMC - PubMed

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