nuclear exosome targeting  (Bio-Techne corporation)

Journal: Molecular cell

Article Title: Interaction profiling identifies the human nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2011.06.028

Figure Lengend Snippet: Figure 2. Identification of the Nuclear Exosome Targeting Complex (A) hMTR4-FLAG SILAC coIP result plotted and labeled as in Figure 1A. High-specificity interactors with log SILAC ratio above 0.5 are indicated in orange. Note disruption of the x axis to accommodate all detected proteins in the plot. (B) Schematic outline of the two different SILAC purification strategies, mixed extracts (ME) and mixed beads (MB), to assay for dynamics of interactions (see the Results for details). SILAC ratios that are constant between experimental strategies signify stable interactions. (C) Interaction dynamics of most prominent hMTR4 binding partners. SILAC ratios are displayed for MB (black) and ME (red) experiments. (D) ZCCHC8-FLAG coIP result plotted and labeled as in Figure 1A. The two groups of high-specificity (log SILAC ratio > 0.5; orange) and high-abundance (signal intensity/MW*106 > 100; blue) interactors. Note disruption of the x axis to accommodate all detected proteins in the plot. (E) Interaction dynamics of most prominent ZCCHC8 binding partners displayed as in (C). (F) The NEXT complex: RBM7-EGFP fusion protein was purified from HEK293 cells using stringent conditions (500 mM NaCl), and eluate was analyzed by SDS-PAGE. The MS identification of Coomassie-stained bands is indicated. Asterisks indicate contaminants.

Article Snippet: the Human Nuclear Exosome Targeting Complex

Techniques: Multiplex sample analysis, Labeling, Disruption, Binding Assay, SDS Page, Staining

Journal: Molecular cell

Article Title: Interaction profiling identifies the human nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2011.06.028

Figure Lengend Snippet: Figure 3. Differential Nuclear Partitioning of Human Exosome Cofactors (A and B) Nuclear-localized hMTR4 and hTRF4-2 accumulate in nucleoli. ZCCHC7 strictly localizes to, and nuclear ZCCHC8 and RBM7 are excluded from, nucleoli. In (A), HeLa cells were transiently transfected with plasmids expressing the indicated proteins as C-terminally EGFP fusions. Fibrillarin staining (red) served as a nucleolar marker and was overlaid with labeling of nuclei by Hoechst stain (blue). In (B), HEK293 Flp-In T-Rex cells stably expressing N-terminally EGFP-tagged proteins were analyzed by live-cell confocal microscopy. Cells were visualized by phase contrast and overlaid with signal from the EGFP fluorescence. (C) ZCCHC8 and ZCCHC7 distribute near hDIS3- and hRRP6-containing low and high molecular weight glycerol gradient fractions, respectively. Western blotting analysis of 5%–40% glycerol gradient fractions of HEK293 cell extract, employing the indicated antibodies (reagents against TRF4-2 and RBM7 were not available). Input corresponds to 5% of the total cell extract.

Article Snippet: the Human Nuclear Exosome Targeting Complex

Techniques: Transfection, Expressing, Staining, Marker, Labeling, Stable Transfection, Confocal Microscopy, High Molecular Weight, Western Blot

Journal: Molecular cell

Article Title: Interaction profiling identifies the human nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2011.06.028

Figure Lengend Snippet: Figure 5. Substrate Preference of NEXT Reflects Its Subnuclear Distribution (A) Western blotting analysis of cell extracts showing protein depletion upon the indicated siRNA administrations. (Top panels) HeLa cells were treated with specific or control (EGFP) siRNAs. Membranes were probed with the indicated antibodies. Anti-actin antibody was used as a loading control. HEK293 cells expressing EGFP-tagged RBM7 (bottom left) or FLAG-tagged hTRF4-2 (bottom right) were treated with the indicated siRNAs. In the EGFP-RBM7 experiment, TEL/AML siRNA was used as control. Protein depletion was assayed using anti-EGFP or anti-FLAG antibodies as indicated. (B) PROMPTs are stabilized in cells depleted for exosome and NEXT complex components. Total RNA from HeLa cells subjected to the indicated siRNA transfections was analyzed by dT-primed RT-qPCR using amplicons for the specific PROMPT regions; ID numbers from left to right: 40-9, -14, -16, -18, -38, -31, -13, -52, -33, and -2b (Preker et al., 2008; Table S14). Data are displayed as mean values normalized to control (EGFP siRNA). All data are normalized to GAPDH RNA as an internal control. Error bars represent standard deviations from biological repeats (n = 3). Note disruption of y axis to accommodate all data in the plot.

Article Snippet: the Human Nuclear Exosome Targeting Complex

Techniques: Western Blot, Control, Expressing, Transfection, Quantitative RT-PCR, Disruption

Journal: Molecular cell

Article Title: Interaction profiling identifies the human nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2011.06.028

Figure Lengend Snippet: Figure 6. Substrate Preference of ZCCHC7 and hTRF4-2 Reflects Their Subnuclear Distributions (A) Schematic representation of the 47S rRNA transcript. Boxes indicate the mature rRNAs 18S, 5.8S, and 28S, which are flanked by the external spacers (ETSs) and separated by the internal spacers (ITSs). (B) Schematic outline of the 50ETS RNA adenylation assay. Adenylated 50ETS RNAs arising from Actinomycin D-induced 47S rRNA degradation are analyzed by RT-PCR using the dT-adaptor oligo, and the indicated 50ETS-1 and adaptor primers. These products are subjected to Southern analysis using the 50ETS-2 probe. (C) Adenylation of 50ETS degradation fragments is compromised upon ZCCHC7 or hTRF4-2 depletion. Southern blotting analysis of RT-PCR products derived as outlined in (B) was performed using the 50ETS-2 hybridization probe. A representative experiment from three repeats is shown. As an internal control, RT-PCR using GAPDH primers was performed. Probe signals were quantified, normalized to GAPDH levels, and plotted relative to EGFP controls. (D) hMTR4 and the core exosome are important for 5.8S rRNA 30 end processing. Total RNA from HeLa cells subjected to the indicated siRNA-mediated knockdowns was used for northern blotting analysis using a probe targeting the ITS2 region (A). Mature 5.8S rRNA was visualized using a radiolabeled DNA oligo probe. Probe signals arising from the 30 end extended species were quantified and plotted relative to EGFP controls. Note that lane 8 is slightly underloaded (Schilders et al., 2007; Tomecki et al., 2010). Error bars and disruption of y axis are as in Figure 5.

Article Snippet: the Human Nuclear Exosome Targeting Complex

Techniques: Reverse Transcription Polymerase Chain Reaction, Southern Blot, Derivative Assay, Hybridization, Control, Northern Blot, Disruption

Journal: Molecular cell

Article Title: Interaction profiling identifies the human nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2011.06.028

Figure Lengend Snippet: Figure 7. Subnuclear Distribution of Human Nuclear Exosome Cofactors Model overview of human nuclear exosome cofactors and their subnuclear localizations as derived from this study. The dually (nucleolar as well as nonnucleolar) localized hMTR4 (structure of its S. cerevisiae homolog [Weir et al., 2010]) is centrally positioned and associates with the nuclear exosome (dashed arrows). In the nonnucleolar part of the nucleus, hMTR4 forms a stable trimeric complex with ZCCHC8 and RBM7. This NEXT complex is excluded from nucleoli, where hMTR4 instead cooperates with putative TRAMP homologous components ZCCHC7 and hTRF4-2. The latter component is also present outside nucleoli.

Article Snippet: the Human Nuclear Exosome Targeting Complex

Techniques: Derivative Assay