PIP2 binds to the largest subunit of Pol I. In order to test the suitability of PLCδ1PH domain for PIP2 detection, we performed ultrastructural immunolabeling and immunofluorescence. (A) Immunogold electron microscopy was carried out using PLCδ1PH domain as a PIP2 sensor. PIP2 was localized at the plasma membrane of HeLa cells as expected. Scale bars: 500 nm. (B) There is no staining in the nucleus when U2OS cells are incubated with PLCδ1PH-Mut domain as a control. Scale bars: 5 µm. (C) PLCδ1PH domain pulled down RPA116 in vitro but the Mut form of PLCδ1PH domain failed to pull down RPA116. (Lane 1, input; lane 2, protein pulled down with Wt PLCδ1PH domain; lane 3, protein pulled down with Mut PLCδ1PH domain.) (D) In vivo, anti-PIP2 antibody showed co-localization with Pol I subunit RPA 116 in nucleoli of U2OS cells. Scale bar: 5 µm. (E) Nuclear extract was incubated with agarose beads coupled to PIP2 in order to pull down proteins interacting with PIP2. Pol I transcription machinery and nucleolar proteins were checked in the pull-down and fibrillarin was found to be present, whereas B23, TAF 95/110 and TBP were absent from the PIP2–protein complex. (Lane 1, input; lane 2, pull-down with PIP2-coupled agarose beads; lane 3, pull-down with control agarose beads; lane 4, marker.) In, input; Ctrl, control.

PIP2 co-localizes with the Pol I transcription factor UBF and the rRNA early processing factor Fib in intact cells. (A) When nucleolar extract was incubated with PIP2-coupled agarose beads, UBF and Fib were found to be present in the PIP2-bound protein complex. [Lane 1, input; lane 2, protein unbound to PIP2-coupled agarose beads (flow-through); lane 3, protein pulled down with PIP2-coupled agarose beads; lane 4, protein unbound to control agarose beads (flow-through); lane 5, protein pulled down with control agarose beads.] In, input; FT, flow-through; Ctrl, control. (B) PLCδ1PH domain, used as a PIP2 marker, co-localized with UBF and Fib in U2OS cells. Scale bars: 5 µm. (C) SIM revealed PIP2 co-localization with UBF and Fib in the subnucleolar components, which can be identified as FC and DFC, respectively. Scale bars: 0.5 µm. (D) IEM precisely distinguished PIP2 co-localization with UBF inside and at the periphery of FC, and with Fib in the DFC of HeLa cells. N, nucleus; NL, nucleolus. Scale bars: 200 nm. (E) Ultrastructural architecture of PIP2 clusters in nucleolar subcompartments by TECNAI G2 20 LaB6 tomography. PIP2 is localized in HeLa cells using a pre-embedding procedure with 0.8 nm immunogold particles pseudocolored in green. The fibrillar center is pseudocolored in yellow, and the dense fibrillar component is in orange.

UBF and Fib bind to PIP2 in vitro. (A) Western blot of purified UBF, Fib, OSH1PH and Imp 5 used in binding assays. (B) PIP2 and PI4P strips were incubated with recombinant UBF, Fib, OSH1PH and Imp 5 proteins for direct binding analysis. (C) Control agarose beads or PIP2-coupled agarose beads were incubated with purified UBF, Fib and Imp 5 proteins to study the direct binding to PIP2. [Lane 1, input; lane 2, protein unbound to PIP2-coupled agarose beads (flow-through); lane 3, protein pulled down with PIP2-coupled agarose beads; lane 4, protein unbound to control agarose beads (flow-through); lane 5, protein pulled down with control agarose beads.] In, input; FT, flow-through; Ctrl, control. (D) Limited protease digestion assays show a difference in the digestion pattern (arrow) of UBF due to a conformational change or specific binding of PIP2. (Lane 1, UBF as an input; lane 2, UBF treated with trypsin; lane 3, UBF treated with trypsin in the presence of PIP2.) (E) Limited protease digestion assays show a difference in digestion pattern of Fib (arrow) due to the conformational change or specific binding of PIP2. (Lane 1, Fib as an input; lane 2, Fib treated with trypsin; lane 3, Fib treated with trypsin in the presence of PIP2.) (F) A footprinting experiment was performed using purified recombinant UBF incubated with 100 ng DAG, IP3 and PIP2. (Lane 1, template incubated with UBF; lane 2, template incubated with UBF in the presence of DAG; lane 3, template incubated with UBF in the presence of IP3; lane 4, template incubated with UBF in the presence of PIP2; lane 5, template only.) UBF binding sites are indicated in the figure. Normalized densitometry plot analysis of the footprint is shown on the left. (G) PIP2 binding to Fib was tested by mobility assays with U6 snRNA where PIP2 association with Fib altered the mobility of RNA, suggesting an additional conformational bend or loop on RNA. [Lane 1, template; lane 2, template incubated with Fib; lanes 3–6, template incubated with Fib in the presence of decreasing amounts of PIP2 (0.1 µg, 0.05 µg, 0.025 µg, 0.0166 µg). Lanes 7–10 are the duplicates of lane 3–6, respectively. See supplementary material Fig. S7 for the entire gel pattern]. Different conformations of RNA–Fib complexes are reflected in the altered mobility of U6 snRNA shown by arrows. Normalized densitometry plot analysis of the gel shift shows Fib complex with U6 snRNA in blue, the Fib and PIP2 complex with U6 snRNA in black at different concentrations of PIP2 and U6 snRNA only as an orange plot. The peak marked as N.S. shows a nonspecific radioactive signal.

PIP2 positive foci co-localize with rRNA nascent transcripts in nucleoli. (A) α-Amanitin treated U2OS cells showed very high co-localization of PIP2 and anti-BrdU positive nascent transcripts. Scale bar: 5 µm. (B) Immunogold detection revealed intermingled clusters and strings of PIP2 and rRNA transcripts in the DFC of HeLa cells. A major portion of Br-rRNA transcripts co-localized with PIP2 molecules, whereas some DFC zones contained only PIP2. NL, nucleolus. Scale bar: 200 nm.