Fig 2: Identification and reconstitution of the ZWC complex. (A) ZC3H4-interacting proteins identified by liquid chromatography–mass spectrometry (LC–MS) analysis. T-REx HEK293 cell lines stably expressing FLAG-ZC3H4 or empty vector were induced with doxycycline and nuclear extracts were prepared and immunoprecipitated with anti-FLAG antibody. Bound proteins were identified by LC–MS analysis. All identified ZC3H4-interacting proteins are listed in Supplementary Table S4. (B) Intracellular interactions between ZC3H4, WDR82 and CK2a. Nuclear extracts prepared from HEK293 cells were immunoprecipitated with an anti-ZC3H4 antibody, and bound proteins were monitored by immunoblotting with the indicated antibodies. (C) SDS-PAGE/Coomassie Blue staining and immunoblot analyses of the reconstituted and purified ZWC complex. The dot indicates a phosphorylated form of CK2a. (D) Cell extracts were prepared from Sf9 insect cells coinfected with combinations of baculoviruses expressing FLAG-tagged (FLAG) or untagged proteins as indicated, and immunoprecipitated with anti-FLAG antibodies (a-FLAG-IP). Proteins were detected by immunoblotting with the indicated antibodies. (E) A model of subunit interactions within the ZWC complex based on the analyses shown in Figure 1D and Supplementary Figure S1C. Note that two copies of CK2a and CK2ß are depicted as present within the complex based on the previously reported heterotetrameric subunit composition of the CK2 complex (20) (left). A schematic diagram of the domains in ZWC complex subunits (right).

Fig 3: CSNK2A1 promotes the proliferation, migration, invasion and EMT of thyroid cancer cells through PI3K–Akt pathway. A, B Knockdown of CSNK2A1 by siRNA, RT-qPCR at mRNA level and western blot at protein level. Effect of CSNK2A1 on the proliferative capacity of thyroid cancer cell lines assessed by clony formation assay (C) and EdU assay(D scale bar:1 mm). Effect of CSNK2A1 on migration and invasive ability of thyroid cancer cell lines assessed by transwell (E scale bar:100 µm) and scratch assays (F scale bar:100 µm). G Effect of circNDST1 on the expression of essential proteins of EMT process in TPC1 cells by Western blot. H Effect of circNDST1 on the expression of essential proteins of PI3K–Akt pathway in TPC1 cells by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are shown as SD ± mean

Fig 4: Direct interaction of the ZWC complex with the Ser5-phosphorylated RNAPII CTD. (A) Test for direct binding of the ZWC complex to unmodified, Ser5-phosphorylated (S5p) and Ser2-phosphorylated (S2p) RNAPII CTD peptides. Biotin pull-down assays employed biotinylated-RNAPII CTD peptides (three copies of YSPTSPS heptad repeats) and purified ZWC complex, and peptide-bound proteins were scored by immunoblotting with the indicated antibodies. (B) Requirement for binding of both ZC3H4 and WDR82 to the S5p RNAPII CTD. The biotinylated S5p RNAPII CTD peptide was tested for binding to the indicated purified complexes and proteins (Supplementary Figure S3B). Note that only the intact ZWC complex was loaded as input (lane 1) and was used for pull-down reactions without peptide (lane 2). Z, ZC3H4; W, WDR82; C, CK2.

Fig 5: The binding assays of hSSRP1 with CK2.A, schematic of full length and truncation of hSSRP1 and CK2. B, coimmunoprecipitation (Co-IP) assays show the interactions between full length and truncation of hSSRP1 with CK2. C, pull-down experiment with GST–hSSRP1-PH3/4, ?CK2a, ?CK2ß,and ?CK2a/CK2ß complex. D, pull-down experiment with GST–hSSRP1-C, ?CK2ß, ?CK2a and?CK2a/CK2ß complex. The input is the total mixture of beads and proteins, and output is the pull-down. The protein gels are stained by Coomassie blue. CK2, casein kinase II; Fl, full length; hSSRP1, human structure-specific recognition protein 1.