Specific threonine-4 phosphorylation and function of RNA polymerase II CTD during M phase progression

Abstract

Dynamic phosphorylation of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 heptad-repeats in the C-terminal domain (CTD) of the large subunit coordinates progression of RNA polymerase (Pol) II through the transcription cycle. Here, we describe an M phase-specific form of Pol II phosphorylated at Thr4, but not at Tyr1, Ser2, Ser5, and Ser7 residues. Thr4 phosphorylated Pol II binds to centrosomes and midbody and interacts with the Thr4-specific Polo-like kinase 1. Binding of Pol II to centrosomes does not require the CTD but may involve subunits of the non-canonical R2TP-Prefoldin-like complex, which bind to and co-localize with Pol II at centrosomes. CTD Thr4 mutants, but not Ser2 and Ser5 mutants, display severe mitosis and cytokinesis defects characterized by multipolar spindles and polyploid cells. We conclude that proper M phase progression of cells requires binding of Pol II to centrosomes to facilitate regulation of mitosis and cytokinesis in a CTD Thr4-P dependent manner.

Figure 1. Thr4-P levels are subject to changes in mitotic cells.

(a) Pol II large subunit (Rpb1) carboxy-terminal domain (CTD) containing heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Consensus repeats have five potential phosphorylation sites. Amino acid residues that differ from the consensus motif are depicted in red. (b) Treatment of HeLa cells with nocodazole induced a slower migrating Thr4-P-specific Pol II00 form. Cells were treated with nocodazole (20 ng/ml) for the indicated time points and extracts of adherent and shake off cells were analyzed by western blotting with mAbs specific for CTD modifications Thr4-P (6D7), Ser2-P (3E10), Ser5-P (3E8), Ser7-P (4E12), or Rpb1 (Pol 3.3). II0 and IIA represent the known hyper- and hypophosphorylated forms of the large subunit Rpb1 of Pol II; II00 represents a new slower migrating form. H3Ser10-P served as a marker for mitotic cells and actin was the loading control. WCE, whole cell extract. a, asynchronous cells. m, mitotic shake off cells. (c) Treatment of HeLa cells with 500 nM okadaic acid (OA) induced the Thr4-P-specific Pol II00 form. Cells were treated with different concentrations of OA for 90 min and whole cell extracts were analyzed by western blotting with antibodies that detected specific CTD modifications, Rpb1 or H3Ser10-P. Tubulin served as the loading control.

Figure 2. Distribution of Pol II populations during mitosis.

(a) The Pol 3.3 monoclonal antibody (mAb) (αRpb1) recognizes a Pol II epitope outside of the CTD and allows the visualization of the distribution of total Pol II in cells (green). Tyr1-P, Ser2-P, Ser5-P, Ser7-P, or Thr4-P-specific mAbs show the abundance and distribution of individual CTD modifications (red) during mitosis. Merged images show localization of signals in the cytoplasm, but also reveal strong enrichment of Thr4-P signals in two distinct foci (red). DAPI: 4′,6-diamidino-2-phenylindole. Representative images of metaphase chromosomes are shown. Data are from three experiments in which at least 100 cells were analyzed and >98% of mitotic cells show these Pol II CTD-P distributions. (b) Images of a Thr4-P-specific mAb (6D7) and DNA (DAPI) in different mitotic human cells (HepG2, H1299). (c) Immunofluorescence images of Thr4-P-specific mAbs of different subclasses (6D7, 1G7, 4H2; red) and a centrosome-specific Ab (γ-tubulin; green) in HeLa cells. Representative images of metaphase chromosomes from three experiments are shown. Signals were merged and quantified using Image J 1.37 V and the plug-in RGB profiler. Line scans were used to measure Thr4-P-specific and γ-tubulin-specific signals. Scale bars, 5 μm.

Figure 3. Thr4 phosphorylated Pol II co-localizes with centrosomes in M phase.

Representative images of a metaphase cells treated with 0.1% Triton X-100 before PFA fixation. (a) HeLa cells were stained with a Thr4-P-specific mAb (6D7, red) and mAb 8WG16 (green) recognizing the CTD of Pol II independently of specific modifications. Co-localisation of Thr4-P and 8WG16 mAbs signals was detected in >98% of mitotic cells. (b) Immunofluorescence image of Rpb3 mAb recognizing the human RNA polymerase II subunit 3 (red) and a centrosome-specific Ab (γ-tubulin; green). Signals from merged images were quantified using Image J 1.37 V and the plug-in RGB profiler. Line scans were used to measure the relative localizations of 8WG16/Rpb3 and Thr4-P-specific signals. (c) Conditional expression of Δ5 mutant in Raji cells. Recombinant polymerase were induced by removal of tetracycline. After 24 h the expression levels of endogenous and recombinant polymerases were analyzed by western blot. The HA mAb allows the visualization of recombinant HA-tagged Pol II (red). Recruitment of Pol II to centrosomes occurs independently of the CTD. Scale bars, 5 μm.

Figure 4. Knockdown of Pol II interactors RUVBL1 and UXT abolishes Thr4-P levels at centrosomes.

(a) The interactome of Pol II. Volcano plot displaying the significant proteins interacting with the recombinant Rpb1 (αHA) when compared to the controls (Pes1 and Bop1). Thresholds: Log 2 Fold change ≥ 5; p-value < 0.01. Indicated are the 12 subunits of Pol II subunits (red) and the 11 subunits of the non-canonical R2TP-Prefoldin-like complex (green). Data based on three biological independent replicates. (b) Western blot analysis of extracts from HeLa cells 48 h after siRNA knockdown of RUVBL1 and UXT. GAPDH3 served as a loading control. (c) Signals for Thr4-P (6D7, red) as well as RUVBL1/UXT (green) co-localized in each phase of mitosis. Representative images of metaphase and anaphase chromosomes are shown. Immunofluorescence images of UXT (green) and Thr4-P-specific (red) mAb in HeLa cells 48 h after siRNA knockdown. >30 cells were analyzed for each experiment; 13% of mitotic cells show loss of the signal for UXT (or RUVBL1) at centrosomes accompanied by the loss of the signal for Thr4-P. Line scans were used to measure the relative localizations of RUVBL1, UXT and Thr4-P-specific signals. Scale bars, 5 μm.

Figure 5. Plk1 and Thr4-P modified Pol II interact in vivo and co-localize at centrosomes in M phase cells.

(a) Co-staining of Plk1 (green) and Thr4-P (6D7; red) in HeLa cells. Representative image of metaphase chromosomes is shown. Line scans measured the relative localization of Plk1 and the Thr4-P-specific signals. Scale bars, 5 μm. (b) HeLa cells were synchronized with nocodazole (20 ng/ml) for 8 h and mitotic cells were collected using the shake off technique. Immunoprecipitation (IP) experiments with antibodies against Plk1 and CTD-Thr4-P (6D7) from extracts of shake off HeLa cells. Immunoprecipitates were analyzed by western blotting with the indicated antibodies. SN, supernatant; IgG, rabbit serum, isotype control. Asterisks, hash-tags and dots indicate bands for Plk1, Thr4-P and heavy chain, respectively. (c) Immunoprecipitated Pol IIA from HeLa cell extracts was used as a substrate for Plk1 and Plk3, and analyzed by Western blotting with mAbs specific for Thr4-P or non-modified CTD (1C7). WCE, whole cell extract.

Figure 6. CTD Thr4 mutants inhibit proper M phase progression.

The phenotype of CTD mutants was studied in human B cell line Raji after expression of CTD mutants. Removal of tetracycline induced expression of (a) recombinant Rpb1 CTD mutants. (b) Immunofluorescence images of γ-tubulin (green) and DNA (DAPI) signals 24 h after induction. (c) Percentage of normal and abnormal cells, with polyploid or lobed nuclei was determined. Data are from three experiments; n = 450, number of cells analyzed for each mutant. Error bars represent standard deviations. (d) The HA mAb allows the visualization of the distribution of recombinant Pol II in cells (red). Arrows indicate a strong enrichment of Pol II in two distinct foci symmetrically to condensed chromatin in metaphase cells. Scale bars, 5 μm.

Figure 7. Model of Pol II function in M phase.

(a) Pol II is recruited to centrosomes of mitotic cells and subsequently phosphorylated at CTD Thr4 residues by Plk1 to promote proper M phase progression. (b) Recruitment of Pol II Thr4/Ala mutant to centrosomes disturbs M phase progression.