The spliceosome is a large ribonucleoprotein complex that catalyzes the removal of introns from RNA polymerase II-transcribed RNAs. Spliceosome assembly occurs in a stepwise manner through specific intermediates referred to as pre-spliceosome complexes E, A, B, B* and C. It has been reported that small molecule inhibitors of the spliceosome that target the SF3B1 protein component of complex A lead to the accumulation of cells in the G1 and G2/M phases of the cell cycle. Here we performed a comprehensive flow cytometry analysis of the effects of isoginkgetin (IGG), a natural compound that interferes with spliceosome assembly at a later step, complex B formation. We found that IGG slowed cell cycle progression in multiple phases of the cell cycle (G1, S and G2) but not M phase. This pattern was somewhat similar to but distinguishable from changes associated with an SF3B1 inhibitor, pladienolide B (PB). Both drugs led to a significant decrease in nascent DNA synthesis in S phase, indicative of an S phase arrest. However, IGG led to a much more prominent S phase arrest than PB while PB exhibited a more pronounced G1 arrest that decreased the proportion of cells in S phase as well. We also found that both drugs led to a comparable decrease in the proportion of cells in M phase. This work indicates that spliceosome inhibitors affect multiple phases of the cell cycle and that some of these effects vary in an agent-specific manner despite the fact that they target splicing at similar stages of spliceosome assembly.

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The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional upregulation of the CDKN1A mRNA and p21WAF1 protein and not to the down regulation of CDK4 or CDK6 by p53-regulated miRNAs.

The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.

21The spliceosome assembles on pre-mRNA in a stepwise manner through five successive 22 pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to 23 generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the 24 spliceosome have been identified and they are cytotoxic. However, little is known about the 25 mechanisms leading to cell death. Activating transcription factor 3 (ATF3) is a transcription 26 factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, 27ATF3 protein levels increased in cultured human and mouse cells in response to cytotoxic levels 28 of the two splicing inhibitors tested: pladienolide B (PB) and isoginkgetin (IGG), that target 29 different pre-spliceosome complexes. Importantly, deletion of ATF3 protected mouse embryonic 30 fibroblasts to these splicing inhibitors. Our results indicate that both splicing inhibitors activate 31 ATF3, and that ATF3 is contributing to the sensitivity of MEFs to these compounds. 32 33 42 sites, the branch sequence, and polypyrimidine tract, leading to the assembly of a functional 43 spliceosome through the E, A, B, B* and C pre-spliceosome complexes[2]. 44 A variety of small molecules interfere with the spliceosome at distinct points in assembly. 45 PB is a spliceosome inhibitor derived from Streptomyces platensis that targets the SF3B1 46 protein. This protein is a component of the U2 snRNP complex so PB prevents the formation of 47 the A complex[3]. IGG is a natural compound derived from Ginkgo biloba, that affects the 48 spliceosome at a subsequent step, preventing the transition from the A to the B complex by 49 inhibiting the stable binding of the U4/U5/U6 tri-snRNP complex[4]. These two compounds 50 affect a different stage of spliceosome formation, however both have been shown to decrease cell 51 viability through uncharacterized mechanisms [5, 6]. 52 Activating transcription factor 3 (ATF3) is a member of the ATF/CREB transcription 53 factor family and is activated in response to a variety of stresses including DNA damage, 54 inflammation, endoplasmic reticulum (ER) stress and oxidative stress [7-10]. ATF3 has been 55 shown to have a variety of functions, one of which is regulating the expression of proapoptotic 56 genes, such as GADD153/CHOP[11]. We sought to determine if ATF3 plays a role in sensitivity 57 to the inhibition of pre-mRNA splicing. Here we report that ATF3 is induced in human and 58 mouse cells and that deletion of ATF3 in mouse embryonic fibroblasts (MEFs) protects cells 59 from the lethal effects of spliceosome inhibitors. This work provides important insight into the 60 mechanisms underlying the cell death induced by spliceosome dysfunction.