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Research Article
Involvement of phosphatidylinositol 4,5-bisphosphate in RNA polymerase I transcription
Sukriye Yildirim, Enrique Castano, Margarita Sobol, Vlada V. Philimonenko, Rastislav Dzijak, Tomáš Venit, Pavel Hozák
Journal of Cell Science 2013 126: 2730-2739; doi: 10.1242/jcs.123661
Sukriye Yildirim
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
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Enrique Castano
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
2Biochemistry and Molecular Plant Biology Department, CICY, Calle 43, No.130, Colonia Chuburná de Hidalgo C.P. 97200, Mérida, Yucatán, México
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Margarita Sobol
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
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Vlada V. Philimonenko
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
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Rastislav Dzijak
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
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Tomáš Venit
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
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Pavel Hozák
1Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics ASCR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic
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  • For correspondence: hozak@img.cas.cz
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  • Fig. 1.
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    Fig. 1.

    PIP2 promotes Pol I transcription in vitro. (A) Run-off transcription reaction showed that addition of anti-PIP2 antibody (clone 2C11, Abcam, Cambridge, UK; 0.8 µg) decreases transcription levels by more than 80%. On the other hand, anti-histone H3 antibody (H0164, Sigma Aldrich, St. Louis, MO, USA; 0.5 µl) had a minor effect on Pol I transcription. (Lane 1, control transcription reaction; lane 2, transcription reaction in the presence of anti-PIP2 antibody; lane 3, transcription reaction in the presence of anti-histone H3 antibody.) The chart shows relative activities (mean±s.e.m.) normalized to an internal DNA control from two independent experiments. (B) The effect of PLC enzyme on in vitro transcription is shown. PLC (100 ng) addition before the addition of nucleotides (PLC B) inhibited Pol I transcription, whereas PLC addition after the addition of nucleotides (PLC A) did not inhibit transcription. (Lane 1, control transcription reaction; lane 2, transcription reaction in the presence of PLC added after nucleotides; lane 3, transcription reaction in the presence of PLC added before nucleotides.) The chart shows relative activities (mean±s.e.m.) normalized to an internal DNA control from two independent experiments (C) Nuclear extracts (NE) were depleted for PIP2 using PLCδ1PH coated beads. As a control, PLCδ1PH-Mut coated beads were used for depletion, since PLCδ1PH-Mut fails in binding to PIP2 (Yagisawa et al., 1998). Transcription intensities were normalized by 700 bp PCR product labeled with [α-32P]. PIP2 depletion by PLCδ1PH domain caused 90% inhibition in transcription, whereas mutant domain did not show such a pronounced inhibitory effect (∼40% inhibition). (Lane 1, non-depleted nuclear extract; lane 2, nuclear extract depleted with PLCδ1PH; lane 3, nuclear extract depleted with PLCδ1PH-Mut domain.) The chart shows relative activities (mean±s.e.m.) normalized to an internal DNA control from two independent experiments (D) PIP2-depleted nuclear extracts supplemented with DAG, IP3, PIP2, PI3P and PI4P were tested for Pol I transcription. PIP2 supplementation resulted in the most dramatic rescue of transcription compared with other compounds tested. (Lane 1, PIP2-depleted nuclear extract; lane 2, PIP2-depleted nuclear extract supplemented with DAG (100 ng); lane 3, PIP2-depleted nuclear extract supplemented with IP3 (100 ng); lane 4, PIP2-depleted nuclear extract supplemented with PIP2 (100 ng); lane 5, PIP2-depleted nuclear extract supplemented with PI3P (100 ng); lane 6, PIP2-depleted nuclear extract supplemented with PI4P (100 ng); lane 7, non-depleted control nuclear extract.) The chart shows relative activities (mean±s.e.m.) normalized to an internal DNA control from two independent experiments. (E) In order to visualize PIP2 during transcription; we used rDNA promoter bound to Dynabeads. GST-tagged Wt or Mut PLCδ1PH domains were purified from bacteria, added to in vitro transcription mixtures and probed with anti-GST antibody. There was no detectable binding of Wt PLCδ1PH domain to the promoter in the presence of ATP solely, and only after the addition of all four rNTPs was binding detected, indicating that PIP2 binds to the promoter region only when transcription is active, in vitro. (Lane 1, PLCδ1PH domain; lane 2, PLCδ1PH-Mut domain; lanes 3 and 4, nuclear extract; lane 5, transcription reaction in the presence of ATP; lane 6, transcription reaction with ATP and PLCδ1PH domain; lane 7, transcription reaction with ATP and PLCδ1PH-Mut domain; lane 8, transcription reaction in the presence of all four rNTPs; lane 9, transcription reaction with rNTPs and PLCδ1PH domain; lane 10, transcription reaction with rNTPs and PLCδ1PH-Mut domain.) (F) In order to prove the presence of PIP2 at the transcription machinery on the promoter during transcription, we used rDNA promoter bound to Dynabeads. PIP2 was found on the rDNA promoter upon the addition of all four rNTPs [N] but not on the control DNA after extracting the lipids with chloroform/methanol/HCl and analyzing them by TLC. (Lane 1, purified PIP2; lanes 2 and 4, lipids from transcription reactions with all four rNTPs; lanes 3 and 5, lipids from in vitro transcription reactions with only ATP added.) Lipids were stained with acidic phosphomolybdate solution.

  • Fig. 2.
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    Fig. 2.

    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.

  • Fig. 3.
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    Fig. 3.

    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.

  • Fig. 4.
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    Fig. 4.

    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.

  • Fig. 5.
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    Fig. 5.

    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.

  • Fig. 6.
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    Fig. 6.

    Model for Pol I transcription. UBF and Pol I interacts with PIP2 during the transcription on the rDNA where PIP2 directs UBF to bind to a more specific site on the promoter compared with its promiscuous binding to the rDNA (transcription assembly). This specific promoter binding occurs in the FC/DFC region. Fib interacts with PIP2 only when RNA is newly synthesized and this interaction takes place in the DFC region close to UBF (transcription initiation/elongation). PIP2 is not involved in further processing of RNA and riboproteins since it is not localized in the GC region where maturation of rRNA takes place. Differences in the hydrophobicity of the complexes may have a role in the formation of subnucleolar structures (processive transcription).

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Keywords

  • Nucleolus
  • Transcription
  • PIP2
  • UBF
  • Fibrillarin

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Research Article
Involvement of phosphatidylinositol 4,5-bisphosphate in RNA polymerase I transcription
Sukriye Yildirim, Enrique Castano, Margarita Sobol, Vlada V. Philimonenko, Rastislav Dzijak, Tomáš Venit, Pavel Hozák
Journal of Cell Science 2013 126: 2730-2739; doi: 10.1242/jcs.123661
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Research Article
Involvement of phosphatidylinositol 4,5-bisphosphate in RNA polymerase I transcription
Sukriye Yildirim, Enrique Castano, Margarita Sobol, Vlada V. Philimonenko, Rastislav Dzijak, Tomáš Venit, Pavel Hozák
Journal of Cell Science 2013 126: 2730-2739; doi: 10.1242/jcs.123661

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