Fig 2: Model: PCBP1 controls exon 5 splicing of the human CKD2 gene transcript via interaction with a recently evolved C-rich PPT. The regions of the mouse and human CDK2 genes that encompass exon 5 and its two flanking exons are shown (not drawn to scale). U: U-rich PPT; C: C-rich PPT. U2AF65 and PCBP1 are RNA binding proteins interacting with U-rich PPT, C-rich PPT, respectively. The mouse U-rich PPT interacts with U2AF65, the canonical PPT binding protein, to ensure that mouse CDK2 exon 4 is spliced to exon 5 constitutively. Human C-rich PPT interacts with PCBP1 proteins to regulate the inclusion of exon 5 in the hCDK2 transcript. The efficiency of AS event is likely to be impacted by the PCBP1 availability and activity (level and modification). When PCBP1 is available, it binds to the C-rich PPT resulting in the inclusion of exon 5. When PCBP1 level/activity is reduced in the cells, exon 5 will be skipped and CDK2 protein expression will be markedly diminished (downward arrow). By this mechanism, the cell cycle can be modulated in response to external or internal cues, through alterations CDK2 expression.

Fig 3: Impact of three additional polypyrimidine binding proteins, hnRNPK, PTB, and RBM39 on CDK2 ex5 splicing. (A) Impact of targeted depletions hnRNPK, PTB, or RBM39 on CDK ex5 splicing. K562 cells were individually transfected with 3 distinct siRNAs targeting the mRNAs that encode each of the indicated proteins. Left: Depletions of individual proteins were confirmed by WB. Ribosomal protein L7a serves as a loading control. Right: The impact of targeted depletions on CDK2 ex5 splicing. Quantification and statistics of the gel are shown at the bottom of the corresponding lanes. n = 3. ∗∗P < 0.01, n.s.: not significant. (B) Impact of dual depletion of PCBPs and PTB on CDK2 ex5 splicing. K562 cells were transfected individually with PCBPs siRNA (siRNA PCBP1/2-2), or PTB siRNA cocktail (mixture of the 3 PTB siRNAs used in Figure 3A), or combination of PCBPs and PTB siRNAs, along with negative control (CTRL), in biological replicates (n = 2). Left: Western blot confirmations of efficient targeted protein depletions. Right: Impact of targeted depletions on CDK2 ex5 splicing as assessed by RT-PCR. Quantification and statistics of the gel were shown at the bottom. ∗∗P < 0.01, ∗∗∗P < 0.001, n.s.: not significant. n = 2.

Fig 4: Impacts of the two major PCBP isoforms, PCBP1 and PCBP2, on CDK2 ex5 splicing. (A) Impact of isoform-specific depletions of PCBP1 and PCBP2 proteins on splicing of exon 5 in the endogenous CDK2 transcript. K562 cells were individually transfected with 2 distinct siRNAs that co-target PCBP1 and PCBP2 mRNAs (siRNA PCBP1/2-1, PCBP1/2-2 as described (19)). The study was done in biological triplicate or with three distinct siRNAs that selectively target PCBP1 (PCBP1-1, PCBP1-2, PCBP1-3), or PCBP2 (PCBP2-1, PCBP2-2, PCBP2-3). Left: Impact of isoform-specific PCBP depletions on cassette exon 5 splicing of the endogenous CDK2 transcript assessed by RT-PCR. Right: Quantification and statistics of the cassette exon 5 splicing data. Summary of the splicing inclusion values are shown on the histogram to the right. ∗∗∗P < 0.001, n.s.: not significant. (B) Impacts of PCBP1 and PCBP2 depletion/repletion on CKD2 ex5 mingene splicing. The minigene (CDK2-C13) plasmid was co-transfected into K562 cells along with a control siRNA (CTRL) or a siRNA co-targeting PCBP1/2 (siRNA PCBP1/2-2 as in (19)) and/or with plasmids expressing recombinant Flag-tagged PCBP1 or PCBP2 (encoded by siRNA-immune mRNAs). The experiment was carried out twice (n = 2). Left: Western blot analysis of protein expression. Labeling of lanes: V (empty vector), CP1 (Flag-PCBP1 expression vector), and CP2 (Flag-PCBP2 expression vector). Western blot assay confirmed siRNA-mediated depletion of endogenous PCBP1 or PCBP2 and expression of the recombinant Flag-PCBP proteins. Right: Impacts of depletion and repletion on CDK2 ex5 minigene splicing. The mean level of exon 5 inclusion (±SD) in each transfection is displayed below the corresponding lane (n = 2). ∗P < 0.05, ∗∗P < 0.01, n.s.: not significant.

Fig 5: Relative impacts of PCBPs and U2AF65 on CDK2 ex5 splicing. (A) Targeted depletion of U2AF65 from K562 cells. The negative control siRNA (Invitrogen, medium GC content) or U2AF65 siRNA (HSS117616, Invitrogen) were transfected into K562 cells in biological duplicates and the level of U2AF65 protein was assessed at 3 days post transfection by Western blot. Loading control corresponds to the ribosomal protein, L7a. (B) U2AF65 depletion fails to significantly impact on CDK2 ex5 minigene splicing. Two CDK2 minigenes, one with the wild type PPT and one with a C13 substitution in the PPT (as in Figure 1B) were co-transfected along with negative control siRNA (Invitrogen, medium GC content) or U2AF65 siRNA (HSS117616, Invitrogen) in biological duplicates. The level of exon 5 inclusion 3 days post transfection is shown below the corresponding lanes of the gel. P values is shown below the gel. (C) Specific impact of PCBP versus U2AF65 on CDK2 ex5 splicing. U2AF65 (3 distinct siRNAs) and PCBPs (siRNA PCBP1/2-1, see Figure 2A) were depleted or co-depleted (siRNA PCBP1/2-1 and PTB siRNA HSS117616, Invitrogen) in K562 cells in biological triplicates and their impacts were assessed on exon 5 splicing of the native CDK2 transcript by RT-PCR. n = 2. Left: RT/PCR analysis of exon 5 inclusion. The positions of the exon 5 inclusion and exclusion bands are indicated to the left of the gel. Quantification of exon 5 inclusion is indicated below the respective lanes. Right: The splicing data are plotted on the adjacent histogram. ∗∗∗P < 0.001. n = 3.