1. Volume 20, Issue 6, Pages 905-915 (December 2005)
Multiple Processing Body Factors and the ARE Binding Protein TTP Activate mRNA Decapping 
Martin Fenger-Grøn, Christy Fillman, Bodil Norrild, Jens Lykke-Andersen 
Molecular Cell 
Volume 20, Issue 6, Pages (December 2005)
DOI: /j.molcel
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2. Figure 1
Identification of Three Proteins Associated with the Decapping Factor hDcp1
(A) Silver-stained SDS-polyacrylamide gels showing proteins that copurify with stably expressed FLAG-tagged hDcp1a (lane 1), or hDcp1b (lane 3), or proteins purified in a similar manner from “parental” cells expressing no FLAG-tagged protein (lanes 2 and 4). The asterisks (∗) show proteins that are either nonspecific or did not reproducibly appear in repeated experiments.
(B) Schematics of Rck/p54, hEdc3, and Hedls proteins (nucleotide sequence accession numbers NP004388, BAB15001, and AAH43616, respectively). Black bars, conserved domains (names given above); gray bars, regions of similarity to orthologs in other organisms. Numbers refer to amino acids (aa).
(C) Western blots of anti-hDcp1a (lane 3) and preimmune (lane 2) immunoprecipitates from RNase-treated HeLa cell extracts. Immunoprecipitates were probed for endogenous Hedls, hEdc3, hDcp1a, and HuR and compared to 10% of the input extract (lane 1). IgG: Immunoglobulin G heavy chain.
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3. Figure 2 Hedls Promotes the Interaction between hDcp2 and hDcp1a
(A) Western blots of anti-FLAG immunoprecipitates from RNase-treated extracts of HEK 293T cells transiently expressing FLAG-tagged hDcp1a (lanes 1–3) or Hedls (lanes 4–6). Precipitates (top panels) and 5% of total extracts (input, bottom panels) were probed for the presence of coexpressed Myc-tagged hDcp2, Hedls, hDcp1a, or hnRNP A1, as indicated.
(B) Western blots of proteins transiently coexpressed in HEK 293T cells from plasmids encoding Myc-Hedls (0 or 0.1 μg, as indicated), Myc-hDcp2 (0 to 1.6 μg, as indicated), FLAG-hDcp1a (0.5 μg), and Myc-hnRNP A1 (0.1 μg). pcDNA3 was added to achieve a total of 2.2 μg of plasmid in each reaction.
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4. Figure 3
Decapping Complex Subunits Localize to PBs, and Their Exogenous Expression Influences PB Morphology
(A) Immuofluorescence assays showing HeLa cells, transiently expressing low amounts of Myc-tagged Hedls, Rck/p54, hEdc3, hDcp1b, or hUpf1 and stained for endogenous hDcp1a and exogenous Myc-tagged proteins, as indicated. Images were merged in the panels on the right (Myc, green; hDcp1a, red).
(B) Immunofluorescence assays showing colocalization between endogenous Hedls (1), hEdc3 (4), and Myc-hDcp1a (2 and 5). Images are merged in 3 and 6 (Hedls and hEdc3, green; Myc-hDcp1a, red).
(C) Same as (A) except that Myc-tagged proteins were overexpressed. Cells were stained for Myc-tagged proteins and endogenous hDcp1a or hXrn1, as indicated.
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5. Figure 4
Hedls Enhances Decapping In Vitro and Triggers Accumulation of Deadenylated Hedls-Associated mRNA When Overexpressed in Cells
(A) Northern blots showing mRNA decay in HeLa Tet-off cells of a β-globin reporter mRNA (β-ARE) that contains an ARE from GM-CSF mRNA in the 3′ UTR, in the presence of exogenous hEdc3, Hedls or no coexpressed protein, as indicated. Time points above the panels refer to minutes after repression of a 6 hr transcriptional pulse. The half-life in each experiment was calculated at ∼65 min. β-GAP, constitutively coexpressed internal control mRNA; β-ARE-A0, product that comigrates with deadenylated β-ARE mRNA (see Figure S5).
(B) Northern blots showing β-ARE and β-ARE-A0 mRNAs that coprecipitate with transiently expressed FLAG-tagged poly-A binding protein (PABP), eIF4E or Hedls, or with no FLAG-tagged protein (−) from extracts of HeLa Tet-off cells that express large amounts of Myc-Hedls (similarly to [A], lane 6). Precipitates (top) and 5% of total extract (bottom) were probed for the presence of β-ARE mRNA.
(C) Thin layer chromatography (TLC) showing the release of m7GDP from a 32P cap-labeled RNA incubated with the indicated proteins. Amount of released m7GDP is given below the panel. A minus sign indicates that m7GDP was not detectable over background. Note that in this assay, hDcp2 alone showed activity below background (lane 2). Representative of five independent experiments with similar results. Origin, origin of loading.
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6. Figure 5
Association and Enhancement of hDcp2 by Activators of mRNA Decay
(A) Western blots for individually expressed Myc-tagged decapping complex subunits that coimmunopurify with FLAG-tagged mRNA decay activator proteins, TTP and hUpf1, from RNase-treated HEK 293 T cell extracts. Precipitates (top panels) are compared with 5% of the input (bottom panels).
(B) Decapping assays using a 32P cap-labeled ARE-RNA (ARE; top panels) or mutant ARE-RNA (ARE-mut; bottom panels) incubated in the presence of the indicated proteins. Amount of released m7GDP is given below the panel. Representative of four independent experiments with similar results. Origin, origin of loading.
(C) Decapping assay as in (B) but using TTP (wt), TTP F126N, or TTP-ΔNTD, as indicated. Representative of three independent experiments with similar results.
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7. Figure 6
Association and Stimulation of a Multimeric Decapping Complex by Activators of mRNA Decay
(A) Schematic depicting observed interactions between proteins in decapping (see Figures S4 and S6). The role in decapping of the complex depicted here is unclear.
(B) Western blots for transiently coexpressed Myc-tagged decapping complex subunits that coimmunopurify with FLAG-tagged mRNA decay activator proteins, TTP and hUpf1, from RNase-treated HEK 293 T cell extracts. Precipitates (top four panels) are compared with 5% of the input (bottom). One asterisk, IgG heavy chain; two asterisks, presumed Hedls degradation product.
(C) Western blots showing coexpressed Myc-tagged proteins that coimmunoprecipitate with FLAG-tagged hEdc3.
(D) Decapping assays using a 32P cap-labeled ARE-RNA incubated in the presence or absence of TTP and the decapping complex (wt) purified through FLAG-hEdc3 ([C], lane 5) or a similarly purified complex (mut) containing the inactive hDcp2 E148Q mutant protein. Amount of released m7GDP is given below the panel. Representative of three independent experiments with similar results. Origin, origin of loading.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Multiple Proteins in Decapping Localize in PBs and Are Recruited to Unstable mRNAs
Proteins in decapping are shown in blue and other PB components in gray. TTP and hUpf1, key components in ARE-mediated decay and NMD, respectively, may communicate with specific decapping subunits, as indicated by arrows. Abbreviations: PTC, premature termination codon; ARE, AU-rich element.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions