p50 protein associates with the C terminus of WRN in vitro. (A) Yeast two-hybrid screening. The WRNCT bait encoding amino acids 949-1401 (∼50 kDa) of the human WRN protein was cloned into pAS2–1 vector and used to screen a human placental cDNA library. The schematic representation of the WRNCT bait shows the C terminus nuclear localization signal (NLS), the conserved RecQ domain (RecQCt), and the putative nucleic acid binding domain (HRD). (B) Retransformation of the yeast expression plasmids to Y190 host strain. pAS2–1, bait vector alone; p50, full-length p50 in pACT2 vector in fusion with GAL4-activator domain. pLAM and pVA3 are human lamin C and murine p53, respectively, cloned into the pAS2–1 vector. (C) In vitro translation combined with immunoprecipitation. WRNCT and full-length p50 cloned into pcDNA3.1-His/Xpress and pcDNA 3.1-His/Myc vectors, respectively, were transcribed/translated in vitro in the presence (WRNCT) or absence (p50) of [³⁵S]methionine. Equal amounts of labeled WRNCT polypeptide were mixed with either control (lanes 1 and 3) or p50-Myc programmed nonradioactive reticulocyte lysates (lanes 2 and 4) and immunoprecipitated by anti-Myc (lanes 1 and 2) or anti-HA (lanes 3 and 4) polyclonal antibodies. The associated polypeptides were resolved by SDS-PAGE and visualized by autoradiography. The lower panel indicates 2.5% of the corresponding mixtures loaded before immunoprecipitation (input). We estimated that 4–5% of the input protein could be coprecipitated with the anti-Myc antibody used.

Ectopic expression of WRN mobilizes p50 from the nucleoplasm to the nucleolus. (A) Exponentially growing asynchronous HeLa cells were transiently transfected with pEGFP vector (a–c), WRNCT (d–f), and full-length WRN cloned in frame to the C terminus of EGFP (g–i) or Myc-tagged full-length p50 (j–l). The cells were imaged by confocal fluorescent microscopy directly visualizing GFP expression (green, a, d, and g) or using indirect immunofluorescence with mouse monoclonal antibody against nucleolin (red, b, e, h, and k) or rabbit polyclonal antibody against the Myc epitope (green, j) followed by Texas Red or FITC-conjugated secondary antibodies, respectively. Merged images show that WRNCT and full-length WRN localize predominantly to the nucleolus (overlapping regions yellow, f and i), whereas p50 localizes to the nucleoplasm excluding the nucleolus (l). (B) Transiently coexpressed WRNCT and p50 colocalize in the nucleolus (d–f). Similar pattern is seen when GFP-tagged full-length WRN is coexpressed with p50-Myc (g–i). GFP-tagged mArf localizes to the nucleolus (green, j) but fails to relocalize p50 (k and l).

Association of WRN with components of the core Pol δ enzyme in vivo. (A) Separate nucleoplasmic (N) and nucleolar (NS) fractions were prepared and immunoprecipitated with normal mouse IgG as control (C) or anti-WRN (WRN) monoclonal antibody. The precipitates were analyzed by Western blotting using anti-p50 antibody (Middle, lanes 1–4). The same blot was reprobed by anti-p125 polyclonal antibody (Top). The bottom panel indicates the respective unprocessed extracts probed with anti-p50 (input, lanes 1–4). (B) Combined nuclear and nucleolar extracts from HeLa cells were immunoprecipitated with normal mouse IgG (C) or anti-WRN monoclonal antibody (WRN) and analyzed by Western blotting with a monoclonal antibody against human PCNA (lanes 1 and 2). Pre-IP inputs for the respective immunoprecipitates are indicated (lanes 3 and 4).

Coimmunoprecipitation of endogenous WRN with p50. Extracts enriched in nuclear and nucleolar proteins were prepared from HeLa cells, SV40-transformed normal human fibroblasts (WI-38), or fibroblast derived from Werner syndrome patients (WS). Aliquots containing approximately 800 μg protein were immunoprecipitated (IP) with a monoclonal antibody against the human WRN protein (WRN) or with normal mouse IgG as control (C). Western blot analysis was performed using anti-p50 (Upper, lanes 1–6) and anti-WRN antibodies (lanes 5 and 6, indicated separately). The lower panel indicates immunoblotting of the respective unprocessed extracts using anti-p50 antibody (input, lanes 1–6).

The components of the endogenous Pol δ core enzyme are recruited to the nucleolus by WRN. HeLa cells were transiently transfected with GFP-tagged cDNAs encoding WRNCT or full-length BLM (green, a and d) and stained with an affinity-purified polyclonal antibody against p50. Exogenously expressed WRNCT, but not BLM, recruits the endogenous p50 to the nucleolus (red, b and e, merged images c and f, respectively). Double immunostaining with anti-Myc and anti-p125 shows that overexpressed p50 (green, g) colocalizes with the endogenous p125 (red, h, merged image yellow, i). Endogenous p50 and p125 staining in untransfected cells are shown in insets (green, g′ and h′). Cotransfection of WRNCT and p50 leads to partial relocalization of p125 to the nucleolus (j–l).