Figure 1.
Effect of CX-4945 on pre-mRNA splicing.
(A) CX-4945 induces expression of an unexpected CK2 α′ transcript, CK2 α′△. RT-PCR corresponding to exons 5–6–7 was performed with total RNA from DMSO- or CX-4945-treated 293T cells. (B) CX-4945 rapidly induces the expression of CK2 α′△. 293T cells were treated with CX-4945 for the times indicated prior to RNA preparation and RT-PCR analysis. GAPDH mRNA was analyzed as a control. (C) Analysis of the DNA sequence of CK2 α′△ revealed that it represents the exon6-skipped product (CK2 α′△exon6) of CK2 α′ pre-mRNA. The normally spliced (CK2 α′) and alternatively spliced (CK2 α′△exon6) products are illustrated schematically.
Figure 2.
The widespread effect of CX-4945 on alternative splicing of numerous genes.
(A) 293T cells were treated with DMSO or 10 µM CX-4945 for 12 hours, and total RNA was prepared. The cDNA was synthesized and further processed for exon array as described in Materials and Methods. All experiments were performed three times, and representative heat maps of selected transcripts from the exon arrays are shown. Selected exons for each mRNA are highlighted with black underlines. (B) RT-PCR analysis was performed for the ELL2, SPAST, QRSL1, PRPSAP2, CPEB1, USP38, TRIP4, CTDSPL2, WDFY1 and ZCCHC2 genes with the same RNA used for the exon arrays. The exonic structure of the RT-PCR products and the exonic location of PCR primers are indicated with tandem rectangles and arrowheads, respectively. PCR products were quantified using ImageJ software, and the ratios of skipped product (skipped/total PCR product) were calculated. The average and SD values were determined from two independent experiments and are presented below the PCR data.
Figure 3.
CX-4945 affects alternative splicing in a CK2-independent manner.
(A) Two other inhibitors of CK2, TBB and TBCA, were also tested to determine whether splicing regulation by CX-4945 is related to CK2 activity. 293T cells were treated with 10 µM CX-4945 or 100 µM TBB or and TBCA for 12 hours. Inhibitory activities of CX-4945, TBB, and TBCA on CK2 were assessed by examination of phosphorylation of AKT(S129), the major substrate of CK2. Total levels of AKT were also monitored as a control. hnRNP A1 was used as a loading control. (B) Alternatively spliced CK2 α′, ELL2, CPEB1, PRPSAP2, and QRSL1 mRNAs were examined by RT-PCR. PCR products of normal and alternatively spliced isoforms are denoted by black arrowheads. An extra PCR product of the QRSL1 gene appeared in TBB-treated samples and is denoted by an asterisk. All experiments were performed twice, and representative data for each are presented.
Figure 4.
CX-4945 modulates SR protein phosphorylation via direct inhibition of Clks.
(A) Total protein extracts from 293T cells treated with CX-4945 (1 and 10 µM) or TG-003 (10 and 50 µM) for 1 hour were separated by SDS-PAGE, and phosphorylated SR proteins were monitored by western blotting using the phosphoSR monoclonal antibody (1H4). SRSF1, SRSF4, SRSF9, and hnRNP A1 proteins were also monitored as controls. Western blotting was performed twice, and representative data are presented. (B) The phosphorylated SR proteins in (A) were quantified, and amounts of each protein relative to those of DMSO-treated samples are shown. The average and SD values were determined from two independent experiments. (C) The potent inhibition of Clks by CX-4945 was observed in in vitro kinase assays conducted by Life Technologies using recombinant human Clk1, Clk2, Clk3, SRPK1, SRPK2, and Ck2 α proteins. The average and SD values were determined from two independent assays. The IC50 values were determined using PRISM software. Detailed experimental procedures are described in the Materials and Methods.
Figure 5.
ATP-competitive inhibition of Clks by CX-4945.
(A) Diagram of the chemical structure of CX-4945 (B) Competition of CX-4945 with ATP for inhibition of Clk2 (C) Structural model of the Clk2 in complex with CX-4945. CX-4945 was predicted to bind to the ATP-binding site of Clk2. (D) The predicted binding mode between CX-4945 and Clk2. Amino acids in the active site are denoted by yellow sticks, and CX-4945 is indicated by green sticks. Hydrogen bonds are denoted as dotted black lines, and oxygen and nitrogen atoms are indicated as red and blue sticks, respectively.