Abstract
Pre-mRNA splicing is indispensable for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest and cell death, which are the reasons for the potent antitumor activity of splicing inhibitors. Here, we found that truncated proteins are involved in cell cycle arrest and cell death upon splicing inhibition. We analyzed pre-mRNAs accumulated in the cytoplasm where translation occurs, and found that a truncated form of the p27 CDK inhibitor, named p27*, is translated from pre-mRNA and accumulates in G2-arrested cells. Overexpression of p27* caused G2-phase arrest through inhibiting CDK–cyclin complexes. Conversely, knockout of p27* accelerated resumption of cell proliferation after washout of splicing inhibitor. Interestingly, p27* was resistant to proteasomal degradation. We propose that cells produce truncated proteins differing functionally from the original proteins via pre-mRNA translation only under splicing-deficient conditions in response to such conditions.
More than 95% of protein-coding genes in humans consist of exons and intervening sequences, namely, introns 1–3. Introns are removed and exons are joined by pre-mRNA splicing to produce mature mRNA: the template for translation. Therefore, defects in splicing machinery cause the accumulation of unspliced or partially spliced mRNAs. If such mRNAs are exported to the cytoplasm and translated into proteins, these proteins would have different amino acid sequences and might have different functions from the original proteins. Such proteins translated from pre-mRNA are supposed to be non-functional or deleterious by inhibiting cellular functions 4 and might cause splicing-related diseases 5–9. To prevent translation from unspliced or partially spliced mRNAs, cells have multiple mRNA quality control mechanisms. For example, the nuclear exosome degrades such unspliced mRNAs 10. Even if unspliced mRNAs escape from this degradation, the export of these mRNAs is strictly prohibited 11–13. If unspliced and partially spliced mRNAs nonetheless leak from the nucleus, they are degraded by nonsense-mediated mRNA decay (NMD) in the cytoplasm 14. These mRNA quality control mechanisms prevent the production of non-functional proteins translated from pre-mRNAs and protect the integrity of the proteome.
Splicing inhibitors, including spliceostatin A (SSA) and pladienolide B (Pla-B), cause G1- and G2/M-phase arrest and cell death 15–18. Such cell cycle arrest and cell death are thought to be the reasons for the antitumor activity of splicing inhibitors. We previously investigated the molecular mechanism of the cell cycle arrest induced by splicing inhibition and found that upregulation of the cyclin-dependent kinase (CDK) inhibitor p27 and a C-terminus-truncated form of p27, named p27*, is one of the causes of G1-phase arrest induced by splicing inhibition 19. Interestingly, p27* is translated from pre-mRNA of CDKN1B (p27) despite mRNA quality control mechanisms 15. In addition, we found that the downregulation of cyclin E1, cyclin E2, and E2F1 is another cause of G1-phase arrest induced by splicing inhibition 20. However, how splicing-deficient cells undergo arrest at the G2/M phase remains completely unclear.
Cell cycle progression is tightly regulated by the kinase activity of CDK–cyclin complexes 21, 22. Among CDKs and cyclins, Cdk1 (also known as Cdc2), cyclin A, and cyclin B are critical regulators of G2/M phase 23–25. The kinase activity of CDK–cyclin complexes is regulated by several mechanisms. The protein levels of cyclins A and B oscillate during the cell cycle. Cyclin A starts accumulating in S phase and suddenly decreases at early M phase 26–30. Cyclin B starts accumulating in G2 phase and decreases at late M phase 24, 31–33. Because cyclin A and B proteins are required for Cdk1 activity, such activity is proportional to the amount of cyclin proteins. In addition to the amount of cyclin proteins, Cdk1 activity is regulated by the phosphorylation status of Cdk1. Cdk1 is phosphorylated at Thr14 and Tyr15 residues in G2 phase, which negatively regulates Cdk1 kinase activity 24, 34, 35. At the end of G2 phase, Cdk1 is dephosphorylated by Cdc25C for activation, and consequently cells enter M phase 24, 36. In addition to the above two mechanisms, CDK inhibitor proteins negatively regulate the kinase activity of CDK–cyclin complexes 22, 37. As mentioned above, p27 is such a CDK inhibitor that controls G1/S transition 38. In fact, p27 is highly expressed in G0/G1 phase and degraded by the ubiquitin–proteasome pathway from S to M phase 39, 40. Therefore, p27 does not appear to control G2/M transition under physiological conditions. However, interestingly, knockout of Skp2, which is a component of SCF (Skp, Cullin, F-box containing complex) ubiquitin ligase for p27 ubiquitylation, causes the accumulation of p27 in G2 phase. The accumulated p27 inhibits M-phase cyclins and consequently causes G2 arrest 40.
In this study, we found that splicing inhibition causes cell cycle arrest and subsequent cell death, and that truncated proteins translated from pre-mRNA contribute to these phenotypes. Furthermore, we revealed that p27*, which inhibits M-phase cyclin and is resistant to proteasomal degradation, is the key factor for the G2-phase arrest upon splicing inhibition.
Results
Splicing inhibition causes cell cycle arrest and cell death
To understand the molecular mechanisms of cell cycle arrest and cell death upon splicing inhibition, we treated HeLa S3 cells with Pla-B and investigated the cell proliferation. Compared with MeOH (the vehicle for Pla-B), 1 ng/ml Pla-B partially inhibited cell proliferation and >2 ng/ml Pla-B completely suppressed it (Fig. 1A). We analyzed cell viability and found that Pla-B treatment had little effect on the cell viability until 24 h and then caused cell death at 48 and 72 h (Fig. 1B). We also investigated when apoptosis starts to occur in Pla-B-treated cells. To this end, we tested whether cleavage of poly(ADP-ribose) polymerase (PARP) is observed in Pla-B-treated cells because cleaved PARP is a useful hallmark of apoptosis 41. We observed PARP cleavage in Pla-B-treated cells at 48 and 72 h, but not at 24 h, which is consistent with the above results (Fig. 1A, 1B, S1A). The p53 tumor suppressor, which is a key factor for apoptosis and cell cycle arrest, is barely expressed in HeLa S3 cells because of its rapid degradation42–44. It is possible that such low expression of p53 affects the timing of onset of apoptosis and cell cycle arrest. To test this hypothesis, we performed the same experiments using breast cancer cell lines: MDA-MB-231 cells and MCF7 cells harboring mutant p53 and wild-type p53, respectively. We observed no conspicuous differences in cell proliferation, cell viability, and PARP cleavage upon Pla-B treatment between the two cell lines, suggesting that Pla-B treatment causes cell cycle arrest at first and then cell death regardless of the existence of wild-type p53 (Fig. S1B–D).
If the cell cycle arrest and cell death are caused by splicing inhibition, cell proliferation should resume after the removal of Pla-B because Pla-B binds to its target protein, SF3B1, non-covalently 45. To test this hypothesis, cells were treated with 3 or 10 ng/ml Pla-B, after which Pla-B was washed out. After washout following 2 h of Pla-B treatment, cell proliferation resumed and almost no dead cells were observed, suggesting that cell cycle progression was arrested only under splicing-deficient conditions (Fig. 1C). Interestingly, however, after washout following 8 h of Pla-B treatment, resumption of cell proliferation was delayed (Fig. 1C). After treatment for 12, 16, and 24 h, the cells did not grow and cell death was observed at 48 and 72 h despite the Pla-B washout (Fig. 1C, 1D). We thus speculated that splicing activity was not restored even after Pla-B washout. To test this hypothesis, we assessed splicing activity in the cells by measuring the relative expression levels of exon–exon junctions and adjacent exons. We found that splicing activity was recovered after washout of Pla-B (Fig. 1E, Fig. S2A, S2B). Notably, the relative expression level of SMEK2 Ex5 was much lower than that of SMEK2 Ex3 (Fig. S2B), which is consistent with previous results indicating that transcription elongation of the SMEK2 gene is inhibited by splicing inhibition46–48. In addition, we investigated whether SF3B1 is phosphorylated after Pla-B washout because SF3B1 is phosphorylated only in active spliceosomes 49, 50. We found that SF3B1 was phosphorylated in the control cells and Pla-B washout cells, but not in the Pla-B-treated cells (Fig. 1F). This also indicates that splicing activity was recovered after the washout of Pla-B. After washout following treatment with 10 ng/ml Pla-B for 16 and 24 h, SF3B1 was not phosphorylated. In such cells, the level of Pol II protein was substantially lower than in control cells, and transcription did not appear to occur, so active splicing might not have occurred (Fig. 1F, S2A). Therefore, we could not observe phosphorylated SF3B1 in such cells. Taking these findings together, we observed cell cycle arrest and cell death even after washout of Pla-B, but splicing activity was recovered in such cells. Therefore, we speculated that mechanisms other than a decrease in gene expression of cell cycle regulators caused by splicing inhibition contribute to the cell cycle arrest and cell death.
Truncated proteins translated from pre-mRNA contribute to cell death and cell cycle arrest
To understand the molecular mechanism of cell cycle arrest and cell death in Pla-B-treated cells, we compared the effect of Pla-B with that of the transcription inhibitor actinomycin D (Act D) or the translation inhibitor cycloheximide (CHX), which inhibit gene expression through different mechanisms. All compounds inhibited cell proliferation (Fig. 2A). Pla-B caused cell death at 48 and 72 h, consistent with the above results (Fig. 2B). Act D appeared to be more toxic than Pla-B, but CHX did not appear to induce cell death even at 72 h (Fig. 2B). These results suggest that CHX induces cell cycle arrest, but not cell death. Next, we compared the effects of CHX and Pla-B on cell cycle progression and found that both of them caused G2/M-phase arrest, but the percentage of G2/M-phase-arrested cells caused by Pla-B was significantly higher than that of CHX-treated cells (Fig. 2C). Because Pla-B and CHX inhibit gene expression but Pla-B caused cell death and G2/M-phase arrest more prominently than CHX, a Pla-B-specific mechanism appeared to cause G2/M-phase arrest and cell death. We assumed that truncated proteins that are translated from pre-mRNA contribute to these phenotypes, and if this is the case, CHX treatment could suppress the effects of Pla-B by inhibiting the production of such truncated proteins. To investigate this hypothesis, we treated the cells with CHX and Pla-B simultaneously and found that CHX treatment indeed suppressed the cell death and G2/M-phase arrest caused by Pla-B treatment (Fig. 2D and 2E), suggesting that truncated proteins translated from pre-mRNA function to cause G2/M-phase arrest and cell death.
Low and high concentrations of Pla-B cause M-phase and G2-phase arrest, respectively
In this study, we decided to investigate the detailed molecular mechanism of G2/M-phase arrest caused by Pla-B treatment. First, we analyzed the effect of Pla-B on cell cycle progression after cell cycle synchronization by a double thymidine block. We found that >2 ng/ml Pla-B treatment caused G2/M-phase arrest by measuring the DNA content of each cell, although 1 ng/ml Pla-B caused partial cell cycle arrest (Fig. 3A, 3B, Fig. S3). Pla-B-treated cells showed an almost identical pattern to control cells until 8 h, suggesting that Pla-B treatment did not affect S-phase progression under these experimental conditions, which is consistent with a previous report 16 (Fig. 3B and S3).
We also investigated the morphology of Pla-B-treated cells because the DNA contents of G2- and M-phase cells are the same, preventing us from distinguishing G2- and M-phase cells based on their DNA content. To this end, we observed the cell morphology and counted the number of cells that were round, a feature of M-phase cells 51. The proportion of round cells among MeOH-treated cells was approximately 40% at 10 h and then decreased (Fig. 3C). Pla-B at 1 ng/ml caused the accumulation of round cells at 14 h, after which the proportion gradually decreased, but ∼30% of cells maintained their round shape (Fig. S4). Upon treatment with 2 or 3 ng/ml Pla-B, round cells had accumulated at 20–36 h (Fig. 3C, S4). In contrast, upon treatment with 5 ng/ml Pla-B, most cells were flat until 24 h, and cells treated with >7 ng/ml Pla-B were flat until 36 h (Fig. 3C, S4). These results suggested that low concentrations of Pla-B caused M-phase arrest, while high concentrations of it caused G2-phase arrest.
In addition to the DNA content and cell morphology, we analyzed the protein levels of cell cycle regulators by immunoblotting upon Pla-B treatment (Fig. 3D and S5). Degradation of cyclin A2, which is a hallmark of M-phase entry, was delayed in cells treated with 2 or 3 ng/ml Pla-B 26, 28, and no degradation of cyclin A2 was observed in cells treated with higher Pla-B concentrations (Fig. 3D and S5), suggesting that a low concentration of Pla-B delays G2/M transition, while a higher concentration of it inhibits this transition. Cyclin B1, which is degraded at late M phase (anaphase) 24, 31–33, was stable in cells treated with >2 ng/ml Pla-B (Fig. 3D and S5), suggesting that a lower concentration of Pla-B causes cell cycle arrest before anaphase. We also examined the phosphorylation status of Cdk1 Y15, which is dephosphorylated at the end of G2 phase and its dephosphorylation is required for the activation of Cdk1 and G2/M transition 36, 52. Dephosphorylation of Cdk1 Y15 was delayed in cells treated with 2 or 3 ng/ml Pla-B, and no dephosphorylation of Cdk1 Y15 was observed in cells treated with higher Pla-B concentrations (Fig. 3D and S5). These results supported the idea that low concentrations of Pla-B cause M-phase arrest, while high concentrations of it cause G2-phase arrest, which is consistent with the above results (Fig. 3B, 3C, S3–S5).
p27* accumulates in G2-phase-arrested cells
The above results indicated that a truncated protein contributes to G2/M-phase arrest (Fig. 2). To identify the truncated protein that is responsible for the G2/M-phase arrest by splicing inhibition, we used our previous data 53. For production of a truncated protein from pre-mRNA, the pre-mRNA should accumulate in the cytoplasm where translation occurs. In our previous study, we found that the splicing patterns of 87 introns are affected in the cytoplasm after splicing inhibition 53. Among these introns, we picked up 13 introns based on Gene Ontology analysis and the length of the estimated truncated protein (Table S1). We performed RT-PCR to investigate whether the 13 introns accumulate in Pla-B-treated cells and found that three introns—RGS2 Ex1-Int4, TBX3 Ex1-Int1, and CDKN1B Ex1-Int1 (p27*)—highly accumulated in Pla-B-treated cells (Fig. S6). We cloned cDNAs to construct expression plasmids of the truncated forms of Rgs2 and Tbx3 and confirmed expression of the truncated proteins (Fig. S7A). Because we had already constructed the p27* expression plasmid 19, we reconfirmed its expression (Fig. S7B). In addition, if antibodies that recognize the N-terminus of truncated proteins were commercially available, we investigated whether endogenous truncated proteins are produced in Pla-B-treated cells. Our results showed that only p27* could be detected, but not Wee1 and Plk2 truncated forms (Fig. S7C). Next, we assessed the effect of the overexpression of Rgs2 truncated protein, Tbx3 truncated protein, and p27* on cell cycle proliferation and cell viability, and found that only p27* inhibited cell proliferation, although it did not affect cell viability (Fig. S7D and E). These results suggest that p27* is the truncated protein responsible for G2-phase arrest caused by splicing inhibition. If this is true, p27* should accumulate in cells under G2 arrest caused by splicing inhibition. Indeed, p27* accumulated in the cells treated with a high concentration of Pla-B that were arrested in G2 phase (Fig. 3D and S5).
Overexpression of p27* causes G2-phase arrest
As mentioned above, p27* accumulated in the G2-arrested cells. Next, we tested whether p27* induces G2-phase arrest. To this end, we established a stable cell line expressing Flag-p27* with expression controlled by a tetracycline-responsive promoter. We confirmed p27* expression only in doxycycline (DOX)-treated cells (Fig. 4A). We observed partial G2/M-phase arrest of DOX-treated cells, suggesting that p27* overexpression causes G2/M-phase arrest (Fig. 4B). We also investigated the expression levels of cyclin A2 in DOX-treated cells. Cyclin A2 was still observed from 14 to 20 h in DOX-treated cells (Fig. 4A), suggesting that a proportion of the Flag-p27*-expressing cells were arrested at early M phase because cyclin A2 is degraded at the end of G2 phase 26, 28. Furthermore, we observed the morphology of the cells at 20 h and found that only ∼5% of the cells were round. This also indicates that most of the arrested cells were arrested in G2 phase, not in M phase (Fig. 4C).
If the accumulation of p27* is the only cause of the G2-phase arrest induced by splicing inhibition, knockdown of p27* should rescue the G2-phase arrest. To test this hypothesis, we used an siRNA against CDKN1B to knock down p27*. Knockdown of p27* was confirmed by immunoblotting (Fig. 4D). At 8 h, most cells were in G2/M phase regardless of treatment with the siRNA and Pla-B (Fig. 4E). Without Pla-B treatment, most cells transitioned to G1 phase at 12 h with or without siRNA treatment. Upon Pla-B treatment, the majority of cells were arrested in G2/M phase, which is consistent with the above results (Fig. 3). However, even after p27* knockdown, the cells were still arrested in G2/M phase at 12 h. In addition, we observed the cell morphology and found that only ∼10% of the cells were round at 12 h (Fig. 4F), suggesting that these cells were arrested in G2 phase, but not in M phase. Therefore, p27* knockdown could not rescue the G2-phase arrest caused by splicing inhibition and, presumably, downregulation of the expression of cell cycle regulator genes also contributes to the G2-phase arrest caused by splicing inhibition.
p27* binds to and inhibits M-phase cyclins
p27* caused partial G2-phase arrest (Fig. 4), and therefore we investigated whether p27* inhibits the kinase activity of M-phase cyclins: Cdk1–cyclin A and Cdk1–cyclin B complexes. To investigate whether p27* bound to M-phase cyclins, we performed immunoprecipitation experiments. Flag-p27* was precipitated using a mouse anti-Flag antibody, and Cdk1 and cyclin B1 were coimmunoprecipitated with Flag-p27* (Fig. 5A, left panel). Because the molecular weights of cyclin A2 and the heavy chain are almost the same, we could not confirm the binding between cyclin A2 and p27*. To confirm the binding, we also performed immunoprecipitation using a rabbit anti-Flag antibody and found that cyclin A2 was coimmunoprecipitated with p27* (Fig. 5A, right panel).
To investigate whether p27* inhibits the kinase activity of the CDK–cyclin complexes, we performed an in vitro kinase assay. Flag-tagged full-length p27 (hereafter called p27 FL) and Flag-p27* were purified and successful purification of the proteins was confirmed by oriole staining and immunoblotting (Fig. S8). Both p27 FL and p27* inhibited the kinase activity of recombinant Cdk1– cyclin A2 and Cdk1–cyclin B1 in a dose-dependent manner (Fig. 5B, 5C). These results suggest that p27* binds to and inhibits M-phase cyclins.
p27* is resistant to proteasomal degradation
To investigate why only p27*, but not p27 FL, was observed in G2-arrested cells, we examined the splicing pattern of the CDKN1B gene. If splicing of CDKN1B pre-mRNA is completely inhibited and spliced mRNA is absent, p27 FL protein, which is translated from spliced mRNA, would not be produced. We tested this hypothesis, but both spliced and unspliced forms of CDKN1B mRNA were observed in the cells, suggesting that both p27 FL and p27* proteins were produced in Pla-B-treated cells (Fig. 6A). Next, we focused on the difference in the C-terminus region between p27 FL and p27*. In the C-terminus of p27 FL, there is a phosphorylation site (threonine 187), the phosphorylation of which is the trigger for ubiquitylation and proteasomal degradation 54. This mechanism keeps the p27 FL protein level low in G2/M phase. Because p27* is a C-terminus-truncated form, p27* lacks this phosphorylation site 15. To investigate whether p27* escapes proteasomal degradation, we evaluated the expression levels of Flag-p27 FL and Flag-p27* in G2/M phase by synchronizing the cell cycle of Flag-p27 FL- and Flag-p27*-expressing cells. Treatment with MG132, a potent proteasome inhibitor, increased the amount of Flag-p27 FL, suggesting that Flag-p27 FL was degraded by the proteasome in G2/M phase (Fig. 6B), which is consistent with a previous report 40. However, the amount of Flag-p27* was not increased by MG132 treatment, but slightly decreased by an unknown mechanism. We also investigated the proteasomal degradation of endogenous p27 FL and p27*. The cell cycle of HeLa cells was synchronized, and the cells were treated with Pla-B and MG132. We found that the amount of endogenous p27 FL was increased by MG132 treatment, but that of p27* was not, suggesting that endogenous p27* is also resistant to proteasomal degradation (Fig. 6C). Because p27* does not have the phosphorylation site for ubiquitylation, we compared the ubiquitylation levels of p27 FL and p27*. To investigate the ubiquitylation level, HEK293T cells were transfected with Flag-p27 FL, Flag-p27*, or vector plasmids. To observe ubiquitylation of p27 FL and p27* clearly, the cells were treated with MG132 and transfected with the HA-Skp2 plasmid 55 because Skp2 protein recruits p27 to SCF ubiquitin ligase. Ubiquitylated proteins were purified from the cells using Tandem Ubiquitin-Binding Entity (TUBE) 56 and analyzed by immunoblotting (Fig. 6D). We observed stronger signals of higher-molecular-weight bands of Flag-p27 FL than that of Flag-p27*. This indicates that p27* was less ubiquitylated than p27 FL, presumably because p27* lacks the phosphorylation site to trigger ubiquitylation. Taken together, these results suggest that p27 FL was degraded by the ubiquitin– proteasome pathway in G2/M phase, but p27* was resistant to degradation; consequently, only p27* accumulated at G2/M phase in Pla-B-treated cells.
p27* was highly stable, and therefore it might remain in the Pla-B washout cells, where it might contribute to the cell cycle arrest after washout of Pla-B (Fig. 1). To test this hypothesis, we investigated the p27* protein level after Pla-B treatment followed by washout (Fig. 6E). As we expected, p27* was still observed even at 72 h. Next, to investigate the effect of the remaining p27* on cell proliferation, we used p27/p27* KO cells 20. Although proliferation of both wild-type and p27/p27* KO cells resumed after Pla-B washout, the recovery of proliferation of p27/p27* KO cells was significantly faster than that of wild-type cells (Fig. 6F). These results suggest that the highly stable truncated protein p27*, which is translated from pre-mRNA, contributes to the cell cycle arrest.
Discussion
Pre-mRNA splicing is one of the most important mechanisms for transcriptome integrity and proteome integrity in eukaryotes. Abnormalities in this process might cause the production of aberrant, non-functional proteins translated from pre-mRNA, which might inhibit functional proteins. To prevent the production of such abnormal proteins, cells have several mRNA quality control mechanisms 10–14: pre-mRNA degradation by the nuclear exosome, nuclear retention of pre-mRNA, and degradation of unspliced and partially spliced mRNAs by NMD. However, in this study, we found that such a truncated protein, p27*, was produced only under splicing-deficient conditions. Why can p27* be produced even though the mRNA quality control mechanisms prevent pre-mRNA translation? We reported that very few selected pre-mRNAs with introns accumulate in the cytoplasm under splicing-deficient conditions53. This indicates that the mRNA quality control mechanisms indeed prevent the accumulation of most pre-mRNAs in the cytoplasm under splicing-deficient conditions. Exceptionally, CDKN1B (p27) pre-mRNA appeared to escape from the mRNA quality control mechanisms. We have revealed that short pre-mRNAs with a weak 5′ splice site tend to escape from the nuclear retention mechanism 53. Because CDKN1B pre-mRNA (5.2 kb) is much shorter than the average length of human genes (27 kb) 57, CDKN1B pre-mRNA might be able to escape from the retention mechanism. Once p27* is produced, p27* appears to remain in splicing deficient cells for a long time. In this study, we observed that p27* is resistant to proteasomal degradation, presumably because it does not possess the site the phosphorylation of which is the trigger for ubiquitination from S to M phase 54. Therefore, p27* is highly stable compared with p27 FL. Another feature might also make p27* a stable protein. p27 FL consists of 198 amino acids, among which exon 1 of CDKN1B encodes 158 15. Most of p27* is thus identical to p27 FL. Therefore, p27* does not appear to be a short peptide, but rather a stable protein. In addition to stability, the high identity between p27* and p27 FL indicates that p27* has physiological function. The CDK inhibitory domain of p27 resides in the N-terminus of p27 37, which is also included in p27*. As a consequence, p27* has the same potent CDK inhibitory activity as p27 FL. Taken together, these features of CDKN1B pre-mRNA and p27* protein make p27* a stable protein with a physiological function.
Why do cells produce p27* under splicing-deficient conditions? We speculate that the CDKN1B gene functions as a sensor for splicing abnormalities. The transcriptome and proteome are perturbed in splicing-deficient cells, and such perturbation might cause splicing-related diseases, including myelodysplastic syndromes and leukemia 5–9. Therefore, the proliferation of splicing-deficient cells might increase the risk of such splicing-related diseases. To reduce this risk, the proliferation of splicing-deficient cells should be inhibited. To inhibit the proliferation of splicing-deficient cells, p27* is a suitable protein because this stable protein with cell cycle inhibitory activity is translated from pre-mRNA only under splicing-deficient conditions. We assume that this system protects the body from splicing abnormality by minimizing the effects of splicing-deficient cells. We also speculate that there are other truncated proteins translated from pre-mRNA to adapt to splicing-deficient conditions. Indeed, in this study, we found that another truncated protein appeared to contribute to cell death upon Pla-B treatment. In a future study, we plan to reveal the entire molecular mechanism by which cells adapt to splicing-deficient conditions using truncated proteins.
The system by which truncated proteins inhibit cell proliferation can be adopted for efforts to achieve tumor suppression. If we inhibit the splicing of cancer cells, the proliferation of such cells should be inhibited by the truncated proteins and downregulation of gene expression. Indeed, Pla-B and SSA have been reported to be potent antitumor reagents 15–18, 58, 59. Although a phase I clinical trial of E7017—a derivative of Pla-B—was discontinued because of serious side effects 60, H3B-8800—another Pla-B derivative—has been investigated in a phase 1/2 study 59, 61. Thus far, the compound has been developed for use on myelodysplasia, acute myelogenous leukemia, and chronic myelomonocytic leukemia patients with spliceosome mutations. This study and future studies regarding truncated proteins translated from pre-mRNA might contribute to the development of a novel antitumor reagent with fewer side effects that targets a broad range of cancers.
In this study, we found that p27* is produced from pre-mRNA in splicing-deficient cells and inhibits cell cycle progression through binding to and inhibiting M-phase cyclins. We believe that these findings deepen our understanding of the mechanisms that protect the body from splicing abnormality and could aid the development of novel anticancer drugs based on splicing inhibitors.
Materials and Methods
Cell culture and synchronization
HeLa S3, HEK293T, MCF7, and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained in 5% CO2 at 37°C. For cell cycle synchronization, the cells were treated with 2 mM thymidine (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 18 h. After treatment, the cells were washed twice with phosphate-buffered saline (PBS) to release them from the thymidine block and then cultured in fresh culture medium for 8 h. The cells were treated with 2 mM thymidine again for 16 h and then washed with PBS twice to release them from the double thymidine block.
Antibodies and reagents
A mouse monoclonal anti-α-tubulin antibody (T6074) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-cyclin A2 (#4656), rabbit polyclonal anti-cyclin B1 (#4138), mouse monoclonal anti-cdc2 (#9116), rabbit polyclonal anti-phospho-cdc2 (Tyr15) (#9111), mouse monoclonal anti-cyclin E1 (#4129), rabbit monoclonal anti-Plk2 (#14812), rabbit monoclonal anti-Wee1 (#13084), and rabbit monoclonal anti-p27 (#3686) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal anti-RNA polymerase II (8WG16) was purchased from BioLegend (San Diego, CA, USA). Mouse monoclonal anti-Sap155 (D-221-3), mouse monoclonal anti-Myc (My3), mouse monoclonal anti-DDDDK (FLA-1), and rabbit polyclonal anti-DDDDK (PM020) antibodies were purchased from Medical and Biological Laboratories (MBL) (Nagoya, Japan). Rabbit polyclonal anti-PARP-1 (H-250) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from GE Healthcare (Chicago, IL, USA).
Doxycycline, puromycin, and G418 were purchased from TaKaRa Bio (Otsu, Japan). Pladienolide B was purchased from Santa Cruz Biotechnology. MG132, actinomycin D, and cycloheximide were purchased from Sigma-Aldrich.
Cell count and cell viability assay
After trypsinization, the numbers of viable and dead cells were counted using trypan blue exclusion and a hemocytometer.
Cell cycle analysis
Cells were fixed in 70% ethanol, rinsed with PBS, and then stained with a solution containing 20 μg/ml propidium iodide (Thermo Fisher Scientific), 0.05% Triton X-100, and 0.1 mg/ml RNase A (Thermo Fisher Scientific). The cell cycle was monitored by the image-based cytometer Tali (Thermo Fisher Scientific).
Morphological observation
Cells were synchronized using thymidine and treated with Pla-B. Morphology of the cells was then observed under an IX73 microscope (Olympus, Tokyo, Japan).
RNA preparation and RT-PCR
Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific), following the manufacturer’s instructions. cDNA was prepared using Primescript Reverse Transcriptase (TaKaRa Bio) and random primers. PCR was performed using TaKaRa ExTaq (TaKaRa). PCR products were analyzed using a 1% agarose gel. To measure splicing activity, we purified nascent RNA using the Click-iT Nascent RNA Capture Kit (Thermo Fisher Scientific). Briefly, cells were treated with 200 µM 5-ethynyl-uridine for 1 h and total RNA was extracted from cultured cells using TRIzol reagent. Labeled RNA was biotinylated by the click reaction and biotinylated RNA was purified using streptavidin beads. For quantitative RT-PCR, cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative RT-PCR and relative quantification analyses were performed with the MX3000P system (Agilent, Santa Clara, CA, USA) using SYBR Green dye chemistry. All primers are listed in Table S2.
Splicing activity (Fig. 1) was calculated using the following formula: Expression level of CDK6 Ex2–3 junction × 2 / (Expression level of CDK6 Ex2 + Expression level of CDK6 Ex3).
Plasmid construction
To construct the RGS2 Ex1-int4 expression plasmid, the DNA fragment of RGS2 Ex1-int4 was amplified by PCR using cDNA prepared from Pla-B-treated cells and the primers RGS2 ATG-Bam F and RGS2 int4STOP-Xho R. The PCR product was digested with Bam HI and Xho I and subcloned into pcDNA3.1/Myc-HIS. To construct the TBX3 Ex1-int1 expression plasmid, the DNA fragment of TBX3 Ex1-int1 was amplified by PCR using cDNA prepared from Pla-B-treated cells and the primers TBX3 ATG-HdIII F and TBX3 int1STOP-Xho R. The PCR product was digested with Hind III and Xho I and subcloned into pcDNA3.1/Myc-HIS. PCR was performed using PrimeSTAR DNA Polymerase (TaKaRa), in accordance with the manufacturer’s instructions. Plasmid transfection was performed using Lipofectamine 3000 Reagent (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions.
Immunoblotting
Cells were directly lysed on plates with 1× SDS-PAGE sample buffer. Proteins were then separated by SDS-PAGE. After electrophoresis, the proteins were transferred onto a PVDF membrane by electroblotting. Following incubation of the membrane with primary and secondary antibodies using standard techniques, protein bands were detected using a NOVEX ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific) on an ImageQuant LAS 4000mini (GE Healthcare).
Stable cell line establishment
To establish HeLa cells expressing Flag-p27 FL or Flag-p27* under the control of tetracycline, a DNA fragment of GFP was amplified by PCR from pcDNA6.2 emGFP using GFP Hind III-Eco RI F and GFP ATTTA R primers. The PCR product was digested with EcoRI and KpnI, and subcloned into pTRE3G-BI (TaKaRa Bio) to construct pTRE3G-BI-GFP. The DNA fragments of Flag-p27 FL and Flag-p27* were amplified from Flag-p27 and Flag-p27* plasmids19, respectively. To amplify Flag-p27, FLAG Hind III-Bgl II F and hp27 cloning R primers were used. To amplify Flag-p27*, FLAG Hind III-Bgl II F and hp27* cloning R primers were used. The PCR products were digested with Bgl II and Not I, and subcloned into pTRE3G-BI-GFP to construct pTRE3G-BI-GFP-FLAG-p27 FL and pTRE3G-BI-GFP-FLAG-p27*. HeLa Tet On 3G cells (TaKaRa Bio) were transfected with pTRE3G-BI-GFP-FLAG-p27 FL or pTRE3G-BI-GFP-FLAG-p27*, and stable clones were selected by puromycin (1 μg/ml) treatment followed by clonal selection. The primers used for plasmid construction are listed in Table S2.
Stable clones were grown in medium containing Tet system-approved fetal bovine serum (TaKaRa Bio), puromycin, and G418. To induce expression, cells were treated for the indicated times with doxycycline (2 μg/ml).
siRNA transfection
Silencer select p27/CDKN1B siRNA (s2837 cat# 4390824) was purchased from Thermo Fisher Scientific. siGENOME Control Pool Non-Targeting #2 (cat# D-001206-14-20) was purchased from GE Healthcare. siRNA transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions.
Immunoprecipitation
Cells were suspended in lysis buffer [25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 2.5 mM EGTA, 1% Nonidet P-40, 10% glycerol, cOmplete Protease Inhibitor cocktail (Sigma-Aldrich), and PhosSTOP (Sigma-Aldrich)] and sonicated for 15 s, and then incubated on ice for 15 min. After centrifugation, cell extracts were incubated with each primary antibody for 1 h at 4°C with gentle agitation and then with Dynabeads Protein G (Thermo Fisher Scientific) for another 1 h. The beads were washed three times with lysis buffer, and then the bound proteins were extracted with 1× SDS-PAGE sample buffer by heating at 95°C for 5 min.
For the purification of ubiquitylated proteins, cells were suspended in lysis buffer [25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 2.5 mM EGTA, 1% Nonidet P-40, 10% glycerol, cOmplete Protease Inhibitor cocktail (Sigma-Aldrich), PhosSTOP (Sigma-Aldrich), and 1 mM N-ethylmaleimide (Nacalai Tesque, Kyoto, Japan)], sonicated for 15 s, and then incubated on ice for 15 min. After centrifugation, cell extracts were incubated with Agarose-TUBE2 (LifeSensors, Malvern, PA, USA) at 4°C for 4 h with gentle agitation. The beads were washed three times with lysis buffer, and then the bound proteins were extracted with 1× SDS-PAGE sample buffer by heating at 95°C for 5 min.
In vitro kinase assay
HEK293T cells were transfected with vector, Flag-p27, or Flag-p27* plasmids19 using Lipofectamine 3000 Reagent, in accordance with the manufacturer’s instructions. Lysates of the transfected cells were prepared using lysis buffer (20 mM HEPES-KCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton, and cOmplete Protease Inhibitor cocktail). Flag-tagged proteins were purified from the cell lysates using a DDDDK-tagged protein purification kit (MBL), in accordance with the manufacturer’s instructions. To evaluate the purification efficiency, the purified proteins were separated by SDS-PAGE, stained with Oriole Fluorescent Gel Stain (Bio-Rad), in accordance with the manufacturer’s instructions, and analyzed by immunoblotting. The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), in accordance with the manufacturers’ instructions. Briefly, the recombinant Cdk1–cyclin A2 or Cdk1–cyclin B1 was mixed with the purified Flag-tagged proteins and then incubated at 25°C for 10 min. Histone H1 and ATP were added to the reaction, followed by incubation at 25°C for 1 h. Next, ADP-Glo Reagent was added to the reaction, followed by incubation at 25°C for 40 min. Finally, Kinase Detection Reagent was added to the reaction, followed by incubation at 25°C for 30 min. Luminescence was measured using a Varioskan Flash (Thermo Fisher Scientific).
Analysis of NGS data
We reanalyzed our previous data (NCBI accession number GSE72156) 53 and calculated the number of amino acids when pre-mRNA is translated from the start codon to the in-frame stop codon in the introns affected by splicing inhibition. We also performed Gene Ontology analysis using AmiGO 2 to select cell cycle regulators 62–64.
Figure S1. Pla-B treatment causes cell cycle arrest and apoptosis. (A) HeLa S3 cells were treated with the indicated concentrations of Pla-B and protein levels of indicated proteins were analyzed by immunoblotting at the indicated time points. (B–D) MDA-MB-231 cells and MCF7 cells were treated with the indicated concentrations of Pla-B for the indicated periods. The number of cells (B) and cell viability (C) were analyzed. Levels of the indicated proteins were analyzed by immunoblotting (D). Error bars indicate s.d. (n = 3).
Figure S2. Splicing activity is recovered after washout of Pla-B. (A, B) HeLa S3 cells were treated with the indicated concentrations of Pla-B for the indicated periods and then washed out. Forty-eight hours after the addition of Pla-B, newly transcribed RNAs were labeled for 1 h and then analyzed by RT-qPCR. Error bars indicate s.d. (n = 3).
Figure S3. Pla-B treatment causes G2/M-phase arrest. Two hours after release from a double thymidine block, synchronized HeLa S3 cells were treated with the indicated concentrations of Pla-B and cell cycle was analyzed at the indicated time points by cytometry. Error bars indicate s.d. (n = 3).
Figure S4. Low doses of Pla-B cause M-phase arrest and high doses of it cause G2-phase arrest. (A, B) Two hours after release from a double thymidine block, synchronized HeLa S3 cells were treated with the indicated concentrations of Pla-B. Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points. Error bars indicate s.d. (n = 3).
Figure S5. p27* accumulates in G2-arrested cells. Two hours after release from a double thymidine block, synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Protein samples were prepared at the indicated time points and the levels of indicated proteins and phosphorylation status of Cdk1 were analyzed by immunoblotting. Protein levels of α-tubulin were analyzed as an internal control.
Figure S6. Pre-mRNAs of RGS2, TBX3, and CDKN1B accumulate in Pla-B-treated cells. Unsynchronized HeLa S3 cells and HeLa S3 cells synchronized at G2/M phase were treated with MeOH or the indicated concentrations of Pla-B for 8 h. Total RNA was purified and the indicated pre-mRNAs were analyzed by RT-PCR.
Figure S7. p27* overexpression causes cell cycle arrest. (A, B) HeLa S3 cells were transfected with expression plasmid of TBX3 Ex1-Int1, RGS2 Ex1-Int4, or CDKN1B Ex1-Int1 (p27*) and expression of the truncated forms was confirmed by immunoblotting. (C) HeLa S3 cells were treated with the indicated concentrations of Pla-B for 8 h and endogenous proteins were detected using the indicated antibodies by immunoblotting. (D, E) HeLa S3 cells were transfected with expression plasmid of truncated forms of TBX3 Ex1-Int1, RGS2 Ex1-Int4, or CDKN1B Ex1-Int1 (p27*) and the number of cells (D) and cell viability (E) were analyzed at the indicated time points. Statistical significance was assessed by the two-tailed t-test (**P < 0.01). Error bars indicate s.d. (n = 3).
Figure S8. Purification of Flag-tagged proteins. (A, B) HEK293T cells were transfected with vector, Flag-p27 FL, or Flag-p27* plasmid, and then protein extracts were prepared. Flag-tagged proteins were purified from the protein extracts using an anti-Flag antibody and then eluted using a Flag peptide. The Flag-tagged proteins were analyzed by oriole staining (A) and immunoblotting (B). Bovine serum albumin (BSA) was also analyzed to estimate the amounts of Flag-tagged proteins.
Acknowledgments
We are grateful to Ms. K. Komori and Mr. K. Kikuchi for technical assistance. We thank Dr. Y. Yoshida (Tokyo Metropolitan Institute of Medical Science) for HA-Skp2 plasmid. We also thank Drs. N. Kataoka and K Fukumura for critical reading of the manuscript. Finally, we thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This research was funded by JSPS KAKENHI (JP21H02423), Takeda Science Foundation, Tamura Science & Technology Foundation, the Kato Memorial Bioscience Foundation, The Ichiro Kanehara Foundation, and Suzuken Memorial Foundation.