Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis

The Xenopus protein Maskin has been previously identified and characterized in the context of its role in translational control during oocyte maturation. Maskin belongs to the TACC protein family. In other systems, members of this family have been shown to localize to centrosomes during mitosis and play a role in microtubule stabilization. Here we have examined the putative role of Maskin in spindle assembly and centrosome aster formation in the Xenopus egg extract system. Depletion and reconstitution experiments indicate that Maskin plays an essential role for microtubule assembly during M-phase. We show that Maskin interacts with XMAP215 and Eg2, the Xenopus Aurora A kinase in vitro and in the egg extract. We propose that Maskin and XMAP215 cooperate to oppose the destabilizing activity of XKCM1 therefore promoting microtubule growth from the centrosome and contributing to the determination of microtubule steady-state length. Further more, we show that Maskin localization and function is regulated by Eg2 phosphorylation

Cooperative Action of Cdk1/cyclin B and SIRT1 Is Required for Mitotic Repression of rRNA Synthesis

<div><p>Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF_I110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD⁺-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAF_I68. Hypoacetylation of TAF_I68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF_I110 is prevented. The results demonstrate that reversible phosphorylation of TAF_I110 and acetylation of TAF_I68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.</p></div

Cdc14B relieves transcriptional repression by dephosphorylating TAF_I110 at threonine 852.

<p>(A) hTAF_I110 is phosphorylated at T852 during mitosis. Tryptic phosphopeptide mapping of TAF_I110. Left: Tryptic phosphopeptide map of Flag-TAF_I110 radiolabeled with extract from mitotic HeLa cells in the presence of [³²P]-ATP. Left: Peptide <b>c</b> (encircled) co-migrates with the synthetic phosphopeptide (SQQHpTPVLSSQPLR). Middle and right: Tryptic phosphopeptide maps of Flag-TAF_I110 radiolabeled with purified Cdk1/cyclin B, and incubated with GST or GST-hCdc14B for 1 h at 37°C in the presence of DMAP. (B) hCdc14B interacts with TAF_I110. GST or GST-hCdc14B were incubated for 4 h at 4°C with ³⁵S-labeled hTAF_I110, hTAF_I68, hTAF_I48, or hTBP. GST-bound proteins were separated by SDS-PAGE and visualized by PhosphorImaging. 10% of input proteins were loaded. (C) TAF_I110 interacts with hCdc14B. HEK293T cells expressing Flag-hTAF_I110 and GFP-hCdc14B were treated with nocodazole (80 ng/ml) for 23 h and released from the nocodazole arrest for 30 min. Flag-hTAF_I110 was immunoprecipitated with M2 antibodies and co-precipitated hCdc14B was monitored on Western blots using anti-GFP antibodies. (D) hCdc14B counteracts Cdk1-mediated mitotic repression of Pol I transcription. Lanes 1, 2: Cdk1/cyclin B was depleted from mitotic extract by pre-incubation with bead-bound p13suc1 and the supernatant was assayed for transcriptional activity. Lanes 3–10: Extract from mitotic HeLa cells was assayed for transcriptional activity in the presence of ATP or AMP-PNP, DMAP, CIAP, or GST-hCdc14B as indicated. The numbers below show the relative amount of run-off transcripts. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s001" target="_blank">S1E Fig</a>. (E) hCdc14B is released from rDNA during mitosis. The bar diagrams present ChIP data showing rDNA occupancy of Cdc14B, UBF and histone H3 in asynchronous cells (as, green bars) and nocodazole-treated mitotic U2OS cells (M, red bars). Bars denote means ±SD from three independent biological replicates (*<i>p</i> < 0.02; ***<i>p</i> < 0.001). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s001" target="_blank">S1F</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s001" target="_blank">S1G</a> Fig, and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s005" target="_blank">S1 Table</a>.</p

Model illustrating inactivation of SL1 and mitotic repression of Pol I transcription.

<p>In interphase cells, TAF_I68 is acetylated by PCAF, which promotes binding of SL1 to the rDNA promoter. At the entry into mitosis, SIRT1 deacetylates TAF_I68, deacetylation weakening the interaction of SL1 with rDNA. Mitotic repression is reinforced by Cdk1/cyclin B-dependent phosphorylation of TAF_I110 at T852, which impairs the interaction between SL1 and UBF. At the end of mitosis, SL1 activity is restored by Cdc14B-mediated dephosphorylation of T852 and <i>de novo</i> acetylation of TAF_I68.</p

Both phosphorylation of TAF_I110 and deacetylation of TAF_I68 are necessary for mitotic inactivation of SL1.

<p>(A) SIRT1 translocates to nucleoli during prophase. Immunostaining of SIRT1, UBF, Pol I, and histone H3 phosphorylated at serine 10 (H3-pSer10) in U2OS cells is shown. Prophase cells are encircled by a dotted line, metaphase cells by a solid line. Representative images out of 100 analyzed mitotic cells are shown. Scale bars, 7 μm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s004" target="_blank">S4 Fig</a>. (B) SIRT1 co-localizes with UBF at the onset of mitosis. Confocal immunofluorescence microscopy of selected cells from (A) showing co-localization of SIRT1 (red) and UBF (green) in prophase, but not in metaphase and interphase cells. Representative images out of 24 mitotic cells are shown. Scale bars, 10 μm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s004" target="_blank">S4 Fig</a>. (C) Pol I transcription is repressed from prometaphase to telophase. FUrd-labeled nascent RNA was visualized by immunofluorescence (FUrd, red), NORs by immunostaining of UBF (green) and chromatin by staining with Hoechst 33342. Mitotic cells are encircled. Representative images out of 200 analyzed cells are shown. Scale bars, 10 μm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s004" target="_blank">S4 Fig</a>. (D) Inhibition of SIRT1 prevents mitotic repression of Pol I transcription in cells expressing TAF_I110/T852A. HeLa cells expressing hTAF_I110/WT or hTAF_I110/T852A were left untreated or treated with NAM (5 mM, 5 h) before pulse-labeling with FUrd. Nascent transcripts were visualized by immunostaining with anti-BrdU antibody (red); NORs were visualized with anti-UBF antibodies (green). Approximately 100 NORscells were analyzed from each cell line; representative images are shown. Scale bars, 10 μm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s004" target="_blank">S4 Fig</a>.</p

SIRT1 deacetylates TAF_I68 in early mitosis.

<p>(A) SIRT1 interacts with SL1. Upper panels: Pull-down experiment using immobilized GST or GST-SIRT1 and the indicated ³⁵S-labeled proteins. Bound proteins were analyzed by gel electrophoresis. The input lanes show 10% of proteins used for pull-down. Lower panel: Western blot showing co-immunoprecipitation of SIRT1 with Flag-hTAF_I68. 2.5% of nuclear extract proteins (Input) and 95% of precipitated proteins were loaded. (B) TAF_I68 interacts with SIRT1, but not with SIRT6 and SIRT7. Flag-hTAF_I68 was co-expressed in HEK293T cells with GFP-tagged SIRT1, SIRT6, or SIRT7. GFP-tagged proteins were bound to GFP-Trap, and co-precipitated Flag-hTAF_I68 was monitored on Western blots using anti-TAF_I68 antibodies (lower panel); SIRTs were monitored with anti-GFP antibodies (upper panel). (C) TAF_I68 is hypoacetylated during mitosis. HeLa-Flag-hTAF_I110 cells were synchronized at G₁/S (G1) by thymidine or in prometaphase (M) by nocodazole treatment. TAF_I68 was immunoprecipitated and analyzed on Western blots using anti-TAF_I68 (10% of IP loaded) or anti-Ac-K-specific antibodies (90% of IP loaded). (D) SIRT1 deacetylates TAF_I68 <i>in vitro</i>. Flag-TAF_I68 expressed and pre-acetylated in Sf9 cells by co-expression with PCAF was incubated with GST-SIRT1 in the absence or presence of 0.5 μM NAD⁺. Acetylation of TAF_I68 was monitored on Western blots using anti-acetyl-lysine antibodies. (See also ref. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.ref021" target="_blank">21</a>]). (E) SIRT1 deacetylates TAF_I68 at early mitosis. Western blot showing acetylation of Flag-TAF_I68 in nocodazole-treated cells cultured in the absence or presence of nicotinamide (+/-NAM) and in cells depleted of SIRT1 (shSIRT1). The input level of SIRT1 and immunopurified Flag-hTAF_I68 is shown. (F) Inhibition of SIRT1 prevents mitotic release of SL1 from rDNA. ChIP showing rDNA promoter occupancy of UBF, SL1 (TAF_I68, TAF_I110, TBP), and Pol I (RPA116) in nocodazole-treated HeLa cells expressing Flag-tagged wildtype (WT, light blue bars) or mutant (T852A) hTAF_I110 (dark blue bars). The graphs depict the relative occupancy in untreated or NAM-treated cells (5 mM, 5 h). The mean values (±SD) from four biological replicates are shown (*<i>p</i>≤ 0.05; **<i>p</i>≤ 0.01).</p

LINC00261 and the Adjacent Gene FOXA2 Are Epithelial Markers and Are Suppressed during Lung Cancer Tumorigenesis and Progression

Lung cancer continues to be the leading cause of cancer-related deaths worldwide, with little improvement in patient survival rates in the past decade. Long non-coding RNAs (lncRNAs) are gaining importance as possible biomarkers with prognostic potential. By large-scale data mining, we identified LINC00261 as a lncRNA which was significantly downregulated in lung cancer. Low expression of LINC00261 was associated with recurrence and poor patient survival in lung adenocarcinoma. Moreover, the gene pair of LINC00261 and its neighbor FOXA2 were significantly co-regulated. LINC00261 as well as FOXA2 negatively correlated with markers for epithelial-to-mesenchymal transition (EMT) and were suppressed by the EMT inducer TGF&beta;. Hierarchical clustering of gene expression data from lung cancer cell lines could further verify the association of high LINC00261/FOXA2 expression to an epithelial gene signature. Furthermore, higher expression of the LINC00261/FOXA2 locus was associated with lung cancer cell lines with lower migratory capacity. All these data establish LINC00261 and FOXA2 as an epithelial-specific marker pair, downregulated during EMT and lung cancer progression, and associated with lower cell migration potential in lung cancer cells

Proteome-Wide Identification of RNA-Dependent Proteins in Lung Cancer Cells

Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells