Pes1 mutagenesis and conditional Pes1 expression. (A) A panel of Pes1 deletion mutants (M1–M8) were constructed. Recombinant Pes1 wt and mutant proteins carry a C-terminal HA-tag. NLS: classical nuclear localization sequence; the N-terminal NLS consists of three overlapping NLS; bip. NLS: six overlapping bipartite NLS; BRCT: BRCT domain; acidic domains: two glutamic acid rich regions; ΨKXE: consensus SUMOylation site; HA: hemagglutinin-tag. Mutant M8 has replaced Lys517 by Arg. (B) Pes1 and its mutants were expressed in rat fibroblasts (TGR-1) stably transfected with the inducible EBV-based vector pRTS-1. pRTS features a bidirectional promoter expressing simultaneously the gene of interest and EGFP. After induction by doxycycline, the vector switches from active silencing to transactivation. (C) EGFP expression was induced in >95% of cells 24 h after addition of doxycycline, as determined by FACS analysis. (D) Western blot analysis of wt or mutant Pes1 proteins in TGR-1 fibroblasts by anti-HA antibodies (3F10). The mock cell line expresses luciferase instead of Pes1-HA. The indicated cell lines were treated with 1 µg/ml doxycycline for 30 h (+) or left untreated (−). Bottom panel shows Ponceau S staining as a loading control.

Cellular localization of wt and mutant Pes1 proteins by indirect immunofluorescence. TGR-1 cells transfected with the indicated constructs were fixed with methanol/acetone after induction of the Pes1-HA alleles with doxycycline for 30 h. The HA-tagged proteins were stained by anti-HA antibodies (3F10), the nuclei were counterstained with DAPI.

Pes1 mutants M1 and M5 inhibit cell proliferation and elicit a reversible cell cycle arrest. (A) Equal numbers of TGR-1 cells stably transfected with the indicated constructs were seeded in multiples in the presence of 1 µg/ml doxycycline. Cells were trypsinized and counted by trypan blue exclusion after 6d. The histogram depicts the cell counts relative to the mock cell line after 6d. Error bars indicate SD. (B) Reversible cell cycle arrest by overexpression of mutants M1 and M5. Stably transfected TGR-1 cell lines were seeded at low density and treated with 1 µg/ml doxycycline for 30 h to induce expression of the specified constructs. Subsequently, the cells were incubated with 100 µM BrdU for 72 h to label proliferating cells. Visible light irradiation in the presence of Hoechst 33 258 selectively kills cells that have incorporated BrdU in their DNA. Arrested cells survive BrdU light treatment and give rise to colonies after withdrawal of doxycycline. Images show representative BrdU light assays conducted with the indicated cell lines.

Dominant-negative Pes1 mutants inhibit pre-rRNA processing. (A) Schematic of eukaryotic pre-rRNA processing. The top diagram shows the primary 47S transcript at the correct scale and the position of the probes used in the northern blots. The lower diagram shows the processing of the pre-rRNA schematically. The effects of M1 and M5 overexpression are indicated. (B) Northern blot for pre-rRNA probed with ITS-2. Total RNA was extracted from subconfluent TGR-1 cells transfected with the indicated constructs after 30 h of induction. Hybridization with a probe against the 18S rRNA confirms equal loading of the blot. (C) Expression of dominant-negative Pes1 mutants impairs formation of mature 28S rRNA. Asynchronously growing TGR-1 cells were treated with doxycycline for 24 h. Cells were pulse labelled with ³²P-orthophosphate for 1 h and chased in regular medium for 0.5 and 3 h. (D) Ratio of 32S/28S rRNA at 3 h after metabolic labelling with ³²P-orthophosphate as described in (C).

Endogenous Pes1 is required for rRNA processing. (A) U2OS cells were transfected at day 0, 1 and 2 with either control or Pes1-specific siRNA. Endogenous Pes1 levels were analysed by western blot analysis 2d after the last transfection. Tubulin is shown as a loading control. (B) U2OS cells were transfected only twice at day 0 and 1 and metabolically labelled with ³²P-orthophosphate for 60 min at day 3. Subsequently, cells were incubated for 3 h in regular culture medium. Labelled rRNAs are indicated.

Dominant-negative Pes1 mutants induce p53 accumulation in proliferating cells but not in quiescent cells. (A) Western blot for endogenous p53 levels. Stably transfected TGR-1 cells were induced at subconfluent density for 30 h with doxycycline. Cell lysates were prepared and analysed by immunoblotting for p53 accumulation using anti-p53 antibodies (Pab-240). n.s.: non specific. (B) Upper panel: western blot as described in (A), but the cells had been serum-starved for 72 h before induction. Lower panel: immunoblot against the HA-tag to confirm the expression of recombinant wt and mutant Pes1. (C) Analysis of the endogenous p53 response to expression of Pes1 wt, and mutants M1 and M5 by indirect immunofluorescence. Asynchronously proliferating and serum-starved (72 h) cells were treated with doxycycline for 24 h to induce the Pes1 constructs. Cells were fixed in methanol and stained against p53 with anti-p53 (Pab122). Cell nuclei were counterstained with DAPI. Representative images are shown. (D) Percentage of p53-positive nuclei in TGR-1 cells upon induction of Pes1 wt, and the dominant-negative mutants M1 and M5. Proliferating and serum-starved cells were analysed by immunofluorescence as described in (C). Numbers of examined cells are indicated in brackets. Error bars indicate SD of the percentage of p53-positive nuclei cells.

Dominant-negative mutants M1 and M5 associate with large pre-ribosomal particles. Western-blot analysis of sucrose gradient fractions of TGR-1 cells expressing Pes1-HA, and Pes1-HA mutants M1, M3 and M5 with an HA-specific antibody. RNA of each fraction was analysed by northern analysis with 28S rRNA specific probe (lower panel).

Incorporation of Pes1 mutants into the PeBoW-complex. U2OS cells stably transfected with the indicated constructs (A–J) were seeded at subconfluent density and treated with doxycycline for 20 h. The lysates were subjected to immunoprecipitation with antibodies either against the HA-tag (3F10), WDR12 (1B8), Bop1 (6H11) or the isotype control coupled to protein G Sepharose beads. The amount of protein loaded in each lane resembles an equivalent amount of total lysate or 20% thereof for the input. ^* indicates cross-reactivity to Ig molecules.

Boundaries and domains important for Pes1 function and PeBoW formation. Pes1 deletions mutants of this work were compared with transposon insertion mutants published by Lapik et al. (15). Deletion mutants conferring a dominant-negative phenotype or affecting formation of PeBoW are indicated.