Fig 1: Effect of PCF11 CID phosphorylation on mRNA export and Pol II interaction. (A) Cytoplasmic and nuclear levels of MYC and TBP mRNA upon WNK463 treatment for 6 h. Data are from two biological repeats. Error bars represent SEM. Data were normalized to siLUC. (B) Quantitation of the percentage of nuclear-localized mRNA over total in fluorescent oligo-dT FISH in cells expressing wild-type or S120A–T121A PCF11 (Supplemental Fig. 7C). The integrated intensities of the nuclear fluorescent signal and the total fluorescent signal were calculated. n = 15. The ratio between the two is presented graphically. Summary data of two biological repeats are shown. Error bars represent SEM. Only cells ectopically expressing PCF11-HA were measured. (C) Detection of MYC mRNA associated with immunoprecipitated wild-type and S120A/T121A PCF11. (D) Co-IP experiment of PCF11 and RNA Pol II. PCF11 immunoprecipitation was followed by Western blot for the detection of Pol II–PCF11 interaction and the comparison of ectopically expressed wild-type and phosphomimetic S120E. (E) RNA Pol II binding to PCF11 CID peptides—unmodified (un) or phosphorylated on S120 (S120ph) or T121 (T121ph)—assayed by Western blot. The first two bands correspond to 5% and 1% of input. A representative blot from three independent experiments is shown. (F) MS analysis of peptide affinity-purified proteins binding to PCF11 CID peptides. The ratio of the spectral index (MIC SIn) of the indicated peptide versus unmodified S120/T121 peptide is shown. Unmodified peptides bind both RPB1 and RPB2 subunits of Pol II in MS, whereas Pol II binding to the same peptide phosphorylated on S120 is ∼10-fold lower, and Pol II binding to the peptide phosphorylated on T121 is entirely lost. Error bars represent standard deviation in three independent experiments. Full data are available in Supplemental Table 3.
Fig 2: WNK1 phosphorylates the PCF11 CID. (A, top) Schematic representation of endogenous PCF11 phosphorylation sites identified in this study (Supplemental Table 2). (Bottom) Schematic representation of the recombinant GST-PCF11 CID. (B) Autoradiography of a recombinant WNK1 kinase domain in vitro kinase assay on the GST-PCF11 CID. MBP was used as a positive control for WNK1 phosphorylation. The upper band corresponds to autophosphorylated WNK1. The lower bands likely correspond to truncated products of the GST-PCF11 CID. (C) Autoradiography of a WNK1 in vitro kinase assay on the wild-type CID and S120A–T121A CID. Coomassie staining shows equal levels of GST-CID loading for wild type and the mutant. (D) Quantitation of kinase assays with the wild-type and S120A–T121A CID. Phosphorylation levels were measured in four independent repeats of the assay. Error bars represent SEM. (E) PhosTag Western blot for the detection of endogenous PCF11 migration upon WNK1 depletion. The same cell extracts treated with λ phosphatase were used as controls. (F) PhosTag Western blots comparing migration of wild-type PCF11-HA and S120A–T121A PCF11-HA in LUC and WNK1-depleted cells. The control lane shows the migration of an unspecific band recognized by the αHA antibody, which provides a positional and loading control.
Fig 3: WNK1 is present in the nucleus and interacts with PCF11. (A) Co-IP experiments of PCF11 and WNK1. Immunoprecipitation of PCF11 (top panel) and WNK1 (bottom panel). Lane 1 corresponds to 5% input. Lanes 2–4 show mock immunoprecipitation using rabbit IgG (negative control). Lanes 2 and 5 show immunoprecipitation with 150 mM NaCl, and lanes 3 and 6 show immunoprecipitation with 250 mM NaCl. Lane 7 shows immunoprecipitation with 150 Mm NaCl after incubation with benzonase to test for nucleic acid dependence. (B) Superresolution microscopy and detection of WNK1 kinase. Pol II staining was used as a control for nuclear localization. The same pattern was observed in all imaged cells. n = 20. Data are from two biological repeats. The same laser intensity was used for all images. (C) Cytoplasmic and nuclear levels of MYC and TBP mRNA in control and WNK1-depleted cells. Data are from biological repeats. Error bars represent standard error of mean (SEM). Data were normalized to siRNA targeting luciferase (siLUC). (D) U6 RNA levels measured with RT-qPCR in nuclear and cytoplasmic extracts.
Fig 4: WNK1 is required for mRNA release from the transcription locus. (A) Schematic representation of the procedure for the detection of heavy chromatin. (B) Detection of the MYC transcription start site (TSS) and 3′ DNA in the heavy (P18) and light (S18) fractions in control cells and WNK1-depleted cells. MYC DNA levels were measured by qPCR. Results were normalized to levels of a gene desert region. (C) Levels of MYC mRNA (measured by RT-qPCR) associated with immunoprecipitated RNA Pol II with or without DNase I treatment. Levels were measured in control cells (siLUC) and WNK1-depleted cells (siWNK1). (D) Levels of MYC mRNA associated with immunoprecipitated Pol II from DNase-treated extracts in control and WNK1-depleted cells with or without empigen treatment. (E) Levels of MYC mRNA associated with immunoprecipitated PCF11 from DNase-treated extracts in control and WNK1-depleted cells with or without empigen treatment. (B–E) Data are from three biological replicates. Error bars represent SEM.
Fig 5: Model. Following cleavage and polyadenylation of the transcript, the CID of PCF11 is transiently phosphorylated by WNK1. This weakens the interaction between the PCF11 CID and the Pol II CTD, leading to the dissociation and release of mRNP from transcription loci. As a result, nuclear export of mRNA is promoted.
Supplier Page from Abcam for Anti-PCF11 antibody