Interaction of Rpp20 and Rpp25 with the P3 domain of human RNase MRP RNA. Biotin-labeled P3 RNA was incubated with in vitro translated Rpp20 and/or Rpp25. Subsequently, P3 RNA-bound material was isolated by precipitation with streptavidin-agarose and analyzed by SDS-PAGE and autoradiography. (A, lanes 1,2) In vitro translated Rpp20 and Rpp25, respectively (100% of the amount used for P3 binding). (Lane 3) Control without P3 RNA. (Lanes 4,5) Precipitated material after incubation with only Rpp20 or only Rpp25. (Lane 6) Precipitated material after incubation with a mixture of Rpp20 and Rpp25. (Lanes 7–11) Precipitated Rpp25 after incubation with P3 RNA in the presence of purified, bacterially expressed GST-Rpp20, GST-Rpp20D36E, GST-Rpp20N40Q, GST-Rpp20G59S, and GST, respectively. (B) Binding of in vitro translated Rpp20 and Rpp25 to biotinylated P3 RNA after incubations with decreasing amounts of Rpp25 (upper panel) or Rpp20 (lower panel) and constant amounts of Rpp20 (upper panel) or Rpp25 (lower panel). (Lanes 1–5) One hundred percent of (diluted) input material (threefold dilution series of the respective proteins in lanes 2–5). (Lanes 6–9) Precipitated material of mixtures of Rpp20 and Rpp25 corresponding to the amounts in lane 1 in combination with that in lanes 2–5.

Subcellular localization of Rpp20 and Rpp25. Immunofluorescence staining of HEp-2 cells with antibodies to Rpp20 (B; mAb 1F11) and Rpp25 (D; polyclonal rabbit serum). A and C show the phase contrast images corresponding to B and D, respectively. The bars in A and C correspond to 10 μm. (E–H) Double immunofluorescence staining of Rpp20 (E, G, H) and Rpp25 (F, G, H). (I–L) Double immunofluorescence staining of Rpp25 (I, K, L) and fibrillarin (J, K, L). (M–P) Double immunofluorescence staining of Rpp25 (M, O, P) and nucleophosmin/B23 (N–P). The images in panels E–P were recorded by confocal microscopy. Bars in panels E–P correspond to 1 μm.

Down-regulation of Rpp20 and Rpp25 expression by RNAi. The expression of either Rpp20 or Rpp25 was inhibited by transfection of HEp-2 cells with corresponding siRNAs. After a second transfection with the same siRNAs at 48 h, cells were lysed at 96 h and the lysates analyzed by Western blotting. An extract from mock transfected cells was generated in parallel as a control. Rpp20 was detected using mAb 1F11, and Rpp25 was detected with a polyclonal rabbit serum against Rpp25. The expression of other RNase MRP/RNase P subunits (Rpp30, Rpp38, and Rpp40) and of an exosome subunit (Rrp4p) in these cells was analyzed using polyclonal rabbit sera specific for each of these proteins.

Rpp20-Rpp25 heterodimerization. Rpp20 and Rpp25 were translated in vitro using a rabbit reticulocyte lysate system. The ³⁵S-labeled proteins were separated by SDS-PAGE and visualized by autoradiography. (A) In vitro translated Rpp20 was incubated with GST-Rpp25 and GST and GST-Rrp41p as controls. GST-tagged and associated proteins were pulled down by glutathione-Sepharose. (Lane 1) In vitro translated Rpp20 (input, 100% of the amount incubated with the GST-tagged proteins). (Lanes 2–4) Radiolabeled protein bound to GST, GST-Rrp41p, and GST-Rpp25, respectively. (B ) In vitro translated Rpp20, Rpp25, or a mixture of these proteins was immunoprecipitated with polyclonal anti-Rpp20 and anti-Rpp25 rabbit sera. (Lanes 1,5) Rpp20 and Rpp25, 10% input. (Lanes 2,3) Immunoprecipitates of Rpp20 with anti-Rpp20 and anti-Rpp25 antibodies, respectively. (Lanes 6,7) Immunoprecipitates of Rpp25 with anti-Rpp25 and anti-Rpp20 antibodies, respectively. (Lanes 4,8) Immunoprecipitates of a Rpp20/Rpp25 mixture with anti-Rpp20 and anti-Rpp25 antibodies, respectively. (C) GST-Rpp25 and GST as a control were incubated with in vitro translated Rpp20 under standard conditions (100 mM KCl, no Triton X-100, lanes 2,3) and in the presence of increasing KCl (upper panel) or Triton X-100 (lower panel) concentrations. (Lane 1) One hundred percent of input material. (Lanes 2–13) GST (even lanes) and GST-Rpp25 (odd lanes) bound material precipitated under the conditions indicated above the lanes. (D) GST pull-down analysis of in vitro translated Rpp25 with equimolar amounts of GST-Rpp20 and GST-Rpp20 amino acid substitution mutants. (Lane 1) In vitro translated Rpp25 (input). (Lanes 2–6) Material bound to GST, GST-Rpp20, GST-Rpp20D36E, GST-Rpp20N40Q, and GST-Rpp20G59S, respectively.

Stoichiometry of Rpp20 and Rpp25 binding to the RNase MRP and RNase P complexes. HEp-2 cells were transiently transfected with constructs encoding VSV-tagged variants of both Rpp20 and Rpp25. As a control, the “empty” VSV-tag vector was used in parallel. Cell lysates were subjected to immunoprecipitation with anti-VSV-tag antibodies, and immunoprecipitated material was analyzed by SDS-PAGE and Western blotting using polyclonal antisera against Rpp30, Rpp40, Rpp20, and Rpp25 as indicated on the left of each panel. Two percent of the input material was loaded in lanes 1–3, and the immunoprecipitated material was loaded in lanes 4–6. (Lanes 1,4) Lysate from cells transfected with the control vector; (lanes 2,5) lysate from cells expressing VSV-Rpp20; (lanes 3,6) lysate from cells expressing VSV-Rpp25. Note that the VSV-tagged proteins have a somewhat higher molecular weight than their endogenous counterparts.

Overexpression of Rpp20 and Rpp25. HEp-2 cells were transfected with constructs encoding GFP (A), GFP-Rpp20 (B), GFP-Rpp25 (C), or both GFP-Rpp20 and VSV-Rpp25 (D) and after 24 h the localization of GFP(-tagged) proteins was visualized by fluorescence microscopy (upper panels). The corresponding phase contrast images are shown in the lower panels. The bar in A corresponds to 10 μm. (E ) The expression of GFP-Rpp20 and VSV-Rpp25 in the transfected cells was analyzed by Western blotting using antibodies to Rpp38, GFP, Rpp20, and Rpp25 (from top to bottom). (Lane 1) Extract from mock-transfected cells; (lanes 2–4) extracts from cells transfected with GFP, GFP-Rpp20, and VSV-Rpp25, respectively; (lane 5) extract from cells cotransfected with GFP-Rpp20 and VSV-Rpp25. The positions of molecular mass markers is indicated on the left (kDa), and the positions of the endogenous and ectopically expressed proteins are marked by arrows on the right.