Pontin is localized in nucleolar fibrillar centers

Abstract

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis.

Fig. 1

Characterization of monoclonal antibodies against Pontin. a Monoclonal antibodies were raised against the full-length recombinant protein and their specificity tested on whole-cell extracts from different cell lines. Only one band of an apparent molecular weight of 50 kD is detected by either one of the antibodies. b To further confirm the antibody specificity, Pontin was detected by the anti-Pontin (5G3-11) antibody in extracts made from cells treated for 48 h or 72 h with anti-Pontin siRNA. A ∼50% reduction at the protein level was observed after the siRNA treatment. In addition, both anti-Pontin antibodies specifically immunoprecipitated Myc-tagged Pontin from cell extracts. c Immunofluorescence labeling of HeLa cells with mouse anti-Pontin monoclonal antibodies (red). DNA counter-stained with DAPI (blue). Prominent dots within the nucleolus were observed in a subset of cells. Staining in the nucleus disappeared after pre-incubation of antibodies with GST-Pontin but not with GST alone showing the specificity of the staining. Scale bar represents 10 μm. d Different regions of Pontin were expressed in E. coli as MBP fusion proteins, purified and used for epitope mapping. The anti-Pontin (3A4-1) antibody interacts with an epitope between the Walker A and B motifs of the protein (amino acids 214-289) while anti-Pontin (5G3-11) antibody binds to an epitope within the C-terminal amino acids 290-456

Fig. 2

Pontin is localized in nucleoli. Double-labeling of HeLa cells with anti-Pontin antibodies 5G3-11 a or 3A4-1 b and anti-fibrillarin, anti-UBF or anti-RNA polymerase I antibodies. The large Pontin dots co-localize with UBF and RNA polymerase I containing dots. In contrast, Pontin revealed only partial co-localization with fibrillarin, which often formed a rim around Pontin dots. Note that in contrast to the three standard nucleolar proteins, Pontin dots appear only in a subset of nucleoli. Scale bar represents 5 μm. c Whole-cell lysate, nucleoplasmic and nucleolar fractions were prepared and used for immunoblotting with the anti-Pontin (5G3-11) antibody. Purity of the nucleolar fraction was verified by detection of a nucleolar marker fibrillarin and absence of the SART3 protein, which is present only in the nucleoplasm and devoid of the nucleolus. SART3 was partially cleaved during the nucleolar preparation and a lower molecular weight band appeared in the nucleoplasmic fraction

Fig. 3

Ultrastructural localization of Pontin. a HeLa cells were synchronized in S-phase and Pontin was detected by the anti-Pontin (5G3-11) monoclonal antibody by means of electron microscopy. In this nucleolar section, Pontin is specifically enriched in a fibrillar center. b Detail of picture a. f fibrillar centers, d dense fibrillar components, g granular components

Fig. 4

Pontin accumulates in the nucleolar dots during S-phase. Pontin was immunodetected by the anti-Pontin (5G3-11) antibody in a unsynchronized cell population or b cells synchronized to S-phase of the cell cycle. Ninety six percent of cells in S-phase accumulated Pontin in the nucleolus. Scale bar represents 10 μm. c Equivalent amounts of proteins from S-phase cells and from unsynchronized cells were loaded in each line (vimentin antibody used as a loading control). No significant differences in protein levels of Pontin were observed between the unsynchronized and synchronized cell populations

Fig. 5

Pontin is involved in the regulation of rRNA transcription. a Pontin was immunoprecipitated using the anti-Pontin (3A4-1) antibody and nuclear proteins detected by immunoblotting. Pol I was specifically precipitated with anti-Pontin antibody but not with a control antibody. b rDNA loci H1 close to the transcription initiation site (952-1,030 bp) and H13 close to the termination site (12,855-12,970 bp) were enriched after chromatin immunoprecipitation with anti-Pontin (5G3-11) or anti-Myc antibodies. Moreover, both regions were specifically amplified after two-step immunoprecipitation with anti-Pontin/anti-Myc or anti-Myc/anti-Pontin antibodies but not with control IgG antibodies showing that Pontin and c-Myc binds to the same regions of rDNA in a complex. Constitutively active GAPDH promoter served as a negative control. c Transcriptional activity of nucleoli containing large Pontin dots is reduced. Newly synthesized RNA (green) was labeled by 10 min incubation with 5-fluorouridine and visualized together with Pontin (red) and fibrillarin (blue) as a marker of nucleoli. In transcriptionally active nucleoli, rRNA signal surrounded Pontin labelling (arrowhead). However, most nucleoli that contained Pontin dots exhibited reduce transcription activity (arrows). Quantification of nucleolar transcription (fibrillarin signal was used as a mask) revealed that transcription activity was reduced by ∼20% in nucleoli where Pontin is sequestered in large dots (average nucleolar fluorescence of transcription signal with standard error of the mean is shown together with number of nucleoli analyzed)