Splicing Of Bcl-x Research Articles

Increased stability of heterogeneous ribonucleoproteins by a deacetylase inhibitor

Splicing factors are often influenced by various signaling pathways, contributing to the dynamic changes of protein isoforms in cells. Heterogeneous ribonucleoproteins (hnRNPs) regulate many steps of RNA metabolism including pre-mRNA splicing but their control by cell signaling particularly through acetylation and ubiquitination pathways remains largely unknown. Here we show that TSA, a deacetylase inhibitor, reduced the ratio of Bcl-x splice variants Bcl-xL/xS in MDA-MB-231 breast cancer cells. This TSA effect was independent of TGFβ1; however, only in the presence of TGFβ1 was TSA able to change the splicing regulators hnRNP F/H by slightly reducing their mRNA transcripts but strongly preventing protein degradation. The latter was also efficiently prevented by lactacystin, a proteasome inhibitor, suggesting their protein stability control by both acetylation and ubiquitination pathways. Three lysines K87, K98 and K224 of hnRNP F are potential targets of the mutually exclusive acetylation or ubiquitination (KAc/Ub) in the protein modification database PhosphoSitePlus. Mutating each of them but not a control non-KAc/Ub (K68) specifically abolished the TSA enhancement of protein stability. Moreover, mutating K98 (K98R) and K224 (K224R) also abolished the TSA regulation of alternative splicing of a Bcl-x mini-gene. Furthermore, about 86% (30 of 35) of the multi-functional hnRNP proteins in the database contain lysines that are potential sites for acetylation/ubiquitination. We demonstrate that the degradation of three of them (A1, I and L) are also prevented by TSA. Thus, the deacetylase inhibitor TSA enhances hnRNP F stability through the KAc/Ub lysines, with some of them essential for its regulation of alternative splicing. Such a regulation of protein stability is perhaps common for a group of hnRNPs and RNA metabolism.

Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5′-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells

Diabetes is a consequence of reduced β-cell function and mass, due to β-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during β-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2β (iPLA2β) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet β-cells. However, chemical inactivation or knockdown of iPLA2β augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2β transgenic mice, whereas islets from global iPLA2β(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2β induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5'-splice site (5'SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA2β/ceramide axis. Exogenous C6-ceramide did not alter Bcl-x 5'SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that β-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2β.

Role of Acinus in regulating retinoic acid-responsive gene pre-mRNA splicing.

Acinus-S' is a corepressor for retinoic acid receptor (RAR)-dependent gene transcription and has been suggested to be involved in RNA processing. In this study, the role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a retinoic acid (RA)-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing of the weak 5' splice site by Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters, while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The RRM domain is necessary for the RA-dependent splicing activity of Acinus and the RA-independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARβ and a RA-independent manner for Bcl-x. Taken together, these studies demonstrate that Acinus functions in both RAR-dependent splicing and RAR-dependent transcription.

Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5' splice site selection.

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5′ splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5′ splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5′ splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5′ splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5′ splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5′ splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator.

The Splicing Factor RBM4 Controls Apoptosis, Proliferation, and Migration to Suppress Tumor Progression

Splicing dysregulation is one of the molecular hallmarks of cancer. However, the underlying molecular mechanisms remain poorly defined. Here we report that the splicing factor RBM4 suppresses proliferation and migration of various cancer cells by specifically controlling cancer-related splicing. Particularly, RBM4 regulates Bcl-x splicing to induce apoptosis, and coexpression of Bcl-xL partially reverses the RBM4-mediated tumor suppression. Moreover, RBM4 antagonizes an oncogenic splicing factor, SRSF1, to inhibit mTOR activation. Strikingly, RBM4 expression is decreased dramatically in cancer patients, and the RBM4 level correlates positively with improved survival. In addition to providing mechanistic insights of cancer-related splicing dysregulation, this study establishes RBM4 as a tumor suppressor with therapeutic potential and clinical values as a prognostic factor.

Bcl-x Pre-mRNA Splicing Regulates Brain Injury after Neonatal Hypoxia-Ischemia

The bcl-x gene appears to play a critical role in regulating apoptosis in the developing and mature CNS and following CNS injury. Two isoforms of Bcl-x are produced as a result of alternative pre-mRNA splicing: Bcl-x(L) (the long form) is anti-apoptotic, while Bcl-x(S) (short form) is pro-apoptotic. Despite the antagonistic activities of these two isoforms, little is known about how regulation of alternative splicing of bcl-x may mediate neural cell apoptosis. Here, we report that apoptotic stimuli (staurosporine or C2-ceramide) reciprocally altered Bcl-x splicing in neural cells, decreasing Bcl-x(L) while increasing Bcl-x(S). Specific knockdown of Bcl-x(S) attenuated apoptosis. To further define regulatory elements that influenced Bcl-x splicing, a Bcl-x minigene was constructed. Deletional analysis revealed several consensus sequences within intron 2 that altered splicing. We found that the splicing factor, CUG-binding-protein-1 (CUGBP1), bound to a consensus sequence close to the Bcl-x(L) 5' splice site, altering the Bcl-x(L)/Bcl-x(S) ratio and influencing cell death. In vivo, neonatal hypoxia-ischemia reciprocally altered Bcl-x pre-mRNA splicing, similar to the in vitro studies. Manipulation of the splice isoforms using viral gene transfer of Bcl-x(S) shRNA into the hippocampus of rats before neonatal hypoxia-ischemia decreased vulnerability to injury. Moreover, alterations in nuclear CUGBP1 preceded Bcl-x splicing changes. These results suggest that alternative pre-mRNA splicing may be an important regulatory mechanism for cell death after acute neurological injury and may potentially provide novel targets for intervention.

Imatinib regulates the alternative pre-mRNA splicing of Bcl-x in K562 cells

Background: The alternative splicing of Bcl-x generates the proapoptotic Bcl-xs protein and the antiapoptotic variant Bcl-xl. Previous studies have demonstrated that some chemotherapeutic agents such as emetine, staurosporine, and epigallocatechin gallate (EGCG) in combination with ibuprofen significantly altered the ratio of the Bcl-x variants Bcl-xs/Bcl-xl in various cell lines, suggesting Bcl-x splicing might be affected by the exogenous stimuli. Objective: We investigated the regulative role of imatinib in the alternative pre-mRNA splicing of Bcl-x in K562 cells and the related mechanism. Methods: Cell proliferation was measured using WST assay kit. Cell apoptosis was assayed using an Annexin V–FITC Apoptosis Detection Kit. RT-PCR and western blot assay was used to analyze the mRNA and protein level of alternative splicing of exon 2 in the Bcl-x gene respectively. Results: Imatinib regulated the alternative splicing in the Bcl-x gene in the K562 cells. In addition, we found that hydroxyurea, another agent for the therapy of CML, could enhance the effect of imatinib on the ratio of the Bcl-xl/Bcl-xs. Moreover, the induction of alternative splicing was correlated with protein phosphatase 1 (PP1). Alternatively, pretreatment with calyculin efficiently blocked imatinib-induced alternative splcing in the K562 cells compared with okadaic acid, which showed an important role of PP1 in regulating imatinib-induced splicing. Conclusion: Imatinib regulates the alternative splicing of Bcl-x in K562 cells, which may be associated with the activation of PP1. Keywords: Alternative splcing, Bcl-x, chronic myelogenous leukemia, imatinib, protein phosphatase 1

Abstract 2081: Mechanisms of the acquisition of anoikis resistance in breast cancer: Lessons from small molecule inhibitors

Abstract The Cox-2 inhibitor Celecoxib has been reported to possess chemopreventive and chemotherapeutic properties, specifically in hormonally-regulated tumors. OSU-03012 is a novel celecoxib derivative, with no COX-2 inhibitory activity, but an order of magnitude greater cell killing ability. We here show that this novel compound has a greater cytotoxicity against a model of anoikis-resistant (AnR) breast cancer. In parallel, anoikis resistance reduces AnR cell sensitivity to the ErbB1/2 dual inhibitor Lapatinib (GW572016, Tykerb, GSK). The two compounds synergize in parental breast cancer cells. Cell killing is achieved partially via induction of ER stress, as measured by eIF2-alpha phosphorylation. We have found that lapatinib resistance may be induced by the aberrant splicing of BCL-X, and that the sensitivity to OSU-03012 is likely due to PTEN dysregulation. In conclusion, these data identify a molecule that may be more efficacious against metastatic tumors, and partially defined its mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2081. doi:1538-7445.AM2012-2081

Identification of a novel cis-element that regulates alternative splicing of Bcl-x pre-mRNA

Alternative splicing plays an important role in the control of apoptosis. A number of genes related to apoptosis undergo alternative splicing. Among them, the apoptotic regulator Bcl-x produces two major isoforms, Bcl-xL and Bcl-xS, through the alternative splicing of exon 2 in its pre-mRNA. These isoforms have antagonistic function in apoptotic pathway; Bcl-xL is pro-apoptotic, while Bcl-xS is anti-apoptotic. The balanced ratio of two isoforms is important for cell survival. However, regulatory mechanisms of Bcl-x splicing remain poorly understood. Using a mini-gene system, we have found that a 105nt exonic region (E3b) located within exon 3 affects exon 2 splicing in the Bcl-x gene. Further deletion and mutagenesis studies demonstrate that this 105nt sequence contains various functional elements which promote skipping of exon 2b. One of these elements forms a stem-loop structure that stimulates skipping of exon 2b. Furthermore our results prove that the stem-loop structure functions as an enhancer in general pre-mRNA splicing. We conclude that we have identified a cis-regulatory element in exon 3 that affects splicing of exon 2 in the Bcl-x gene. This element could be potentially targeted to alter the ratio of Bcl-xL and Bcl-xS for treatment of tumors through an apoptotic pathway.

Alternative splicing of apoptosis-related genes in imatinib-treated K562 cells identified by exon array analysis

Imatinib is the therapeutic standard for newly diagnosed patients with chronic myeloid leukemia (CML). In these patients, imatinib has been shown to induce an apoptotic response specifically in cells expressing the oncogenic fusion protein BCR-ABL. Previous studies in our lab revealed that imatinib-induced apoptosis in K562 cells involves a shift in production of Bcl-x splice isoforms towards the pro-apoptotic Bcl-xS splice variant. Here, we report the findings from our subsequent study to identify other apoptosis-related genes that are differentially spliced in response to imatinib treatment. Gene expression profiling of imatinib-treated K562 cells was performed by the Affymetrix GeneChip® Human Exon 1.0 ST array, and differences in exon-level expression and alternative splicing were analyzed using the easyExon software. Detailed analysis by reverse transcription-PCR (RT-PCR) and sequencing of key genes confirmed the experimental results of the exon array. Our results suggest that imatinib treatment of K562 cells causes a transcriptional shift towards alternative splicing in a large number of apoptotic genes. The present study provides insight into the molecular character of apoptotic leukemia cells and may help to improve the mechanism of imatinib therapy in patients with CML.

Pharmacologic Doses of Amiloride Preferentially Induce Apoptosis and Growth Inhibition of Flt3-ITD Mutation Positive Acute Myeloid Leukemia Cell Lines

Abstract 5004 Introduction:Amiloride is an FDA approved potassium-sparing diuretic which targets Na+/H+ exchanger isoform 1 (NHE1). NHE1 is responsible for the regulation of the intracellular pH, as well as cell-cycle and apoptosis. In supra-pharmacologic concentrations, amiloride non-specifically inhibits protein kinases. Recent study demonstrated that proapoptotic effect of amiloride in CML cell lines is linked to the modulation of the alternative splicing of Bcl-x, HIPK3, and BCR/ABL genes and is independent of pHi.Here, we demonstrate that pharmacologic doses of amiloride preferentially induce growth inhibition, cell cycle arrest and apoptosis in Flt3-ITD positive acute myeloid leukemia cell lines as compared to Bcr-Abl positive leukemia cell line. Our data suggests that amiloride may have an effect on Flt3 signaling and that its treatment potential for Flt3-ITD positive acute myeloid leukemia needs to be explored. Methods:MV4-11, MOLM13 and K562 cells lines in log-phase growth were used for the experiments. Analysis of the baseline Flt3 expression and phosphorylation status was assessed via Flt3 immunoprecipitation and Western blotting for Flt3 and phosphotyrosine. Cells were incubated with various amiloride concentrations; equal volume dilutions of DMSO were used for control. Cell counting and trypan blue exclusion viability was performed on TC10 Bio-Rad automated cell counter. The cell cycle analysis was performed applying propidium iodide staining. To assess for apoptosis and cell death, we used annexin V/PI staining kit and flow cytometry. Results:MOLM13 and MV4-11 cell lines carry activating Flt3-ITD mutation. We confirmed the expression and constituative activation of Flt3 in MOLM13 and MV4-11 cells with Western blotting. Flt3 protein was not detectable in K562 cell line. Amiloride at 0.025 mM and 0.05 mM completely inhibited the growth of MV4-11 cells after 24 hrs of treatment with no significant increase in total or live cell numbers at 72 hrs, but only mildly affected K562 cell proliferation. While the above amiloride concentrations caused cell death in MV4-11 and MOLM13 cell lines, there was no increased cell death in K562 cells.Incubation of MOLM13 and MV4-11 cell lines with 0.05 mM amiloride for 20 hrs induced cell cycle arrest. In MV4-11 cell line, the proportion of S phase cells after amiloride treatment was 15.4% (SD=5.4%) as compared to 31.3% (SD=1.4%) in control. MOLM13 cell line demonstrated 15.3% (SD=4.7%) of cells in S after amiloride treatment as compared to 35.3% (SD=2.4%) cells in S phase in control treatment. In K562 cell line, there was less effect with 52% (SD=4.2%) of cells in S phase in control as compared to 37% (SD=8.9%) in amiloride treatment.MV4-11 and MOLM 13 cell lines were more sensitive than K562 cells to amiloride induced apoptosis with 28.8% (control 12.7%) of MV4-11 cells, 11.4% (control 7.4%) of MOLM13 cells, and 11.4% (control 8.6%) of K562 cells being apoptotic after 20 hr treatment with 0.05mM amiloride. At 72 hrs of amiloride treatment 34% (control 1.5%) of MV4-11 cells, 17% (control 5%) of MOLM13 cells and 11% of K562 cells (control 8.9%) were apoptotic. Amiloride had similar effect on the number of dead cells with no increase in total cell death in K562 cell line. Upon treatment with increasing amiloride concentrations, there was dose-dependent increase in cell death and apoptosis in all three cell lines with K562 line showing relative resistance to amiloride. Discussion:Our results demonstrate that amiloride induces cell cycle arrest and inhibits proliferation of Flt3-ITD positive cell lines MV4-11 and MOLM13 as well as K562 cell line at a pharmacologic concentration of 0.05 mM. Both, cell cycle arrest and antiproliferative effect are more pronounced in Flt3-ITD positive cells lines while it is mild in Bcr-Abl positive K562 cell line. Pharmacologic doses of amiloride induce cell death and apoptosis in Flt3-ITD positive cell lines but not in K562 cell line. Both, Bcr-Abl and Flt3 signaling stimulates proliferation and inhibits apoptosis in myeloid leukemia cells. Our study suggests that amiloride may induce cell cycle arrest and apoptosis via modulating Flt3 signaling cascade. We are currently investigating the effects of amiloride on Flt3 phosphorylation. In conclusion, our data suggests that amiloride presents a potential treatment option for Flt3-ITD positive acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.

Abstract 4771: The oncogenic kinase Bcr-Abl regulates splicing of Bcl-x trough a quaternary complex coordinated by Nck-beta and Sam68 adapter proteins

Abstract Alternative splicing plays a key role for generating protein diversity and contribute to essential biological processes among them differentiation, cell cycle regulation and apoptosis. Alterations in alternative splicing was identified in several human diseases, including cancer. Bcl-x is a Bcl-2 family member which is alternatively spliced in two distinct transcripts generating two different proteins: Bcl-xL (Long) and Bcl-xS (Short), with antagonistic functions. The long isoform inhibits apoptosis, in contrast the short one activates it. Bcl-xL is highly expressed in different types of cancer. CML is a myeloproliferative disorder characterized by a reciprocal chromosomal translocation t(9;22), that generate Bcr-Abl chimeric protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cell proliferation, alteration in cell-cell and cell-matrix adhesion and resistance to apoptosis. It has been reported that in CML Bcl-x isoforms ratio is altered in favor of the anti-apoptotic one (Bcl-xL).We investigated the involvement of Bcr-Abl in regulating splicing events affecting Bcl-x pre-mRNA occurring in CML. By proteomic approach, based on strategy using GST-Pull Down assay, we attempted to gain insight into functional interactions occurring among the oncogenic kinase Bcr-Abl and RNA-binding proteins, identifying physical interactions among the adapter protein Nck-beta, the spliceosome ribonucleoprotein hnRNPA1 and Sam68 which are assembled to form a functional quaternary complex. These interactions were further confirmed by immunofluorescence staining showing co-localization of the proteins within peculiar nuclear splicing regions (nuclear speackles). Using RNA Pull Down assays we detected that the quaternary complex was able to specifically bind BCL-xL mRNA. Using several specific tyrosine kinase inhibitor such as BMS-354825, AMN-107 or STI-571, we identified a key role of BCR-ABL phosphorylation on integrity of the multimeric complex. Furthermore, STI-571 impairs the interaction between quaternary complex and Bcl-xL mRNA. As observed also for other spliced genes like MCL1, PP1γ; PASG, VRK and CENT1. The Bcr-Abl mediated Bcl-x splicing was also confirmed by quantitative PCR and immunofluorescence analysis. Bcr-Abl leads to increased Bcl-xL mRNA expression as proved either by Bcr-Abl ectopic overexpression or by a Bcl-x minigene assay. In conclusion, these data indicate that phosphorilated Bcr-Abl is part of a complex affecting Bcl-x alternative splicing, increasing the anti-apoptotic isoform in respect to the pro-apoptotic one. These data represent the first original report of a direct involvement of Bcr-Abl in splicing events, thus opening new perspectives in CML therapeutic treatment field. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4771. doi:10.1158/1538-7445.AM2011-4771

An Alternative Splicing Network Links Cell-Cycle Control to Apoptosis

Alternative splicing is a vast source of biological regulation and diversity that is misregulated in cancer and other diseases. To investigate global control of alternative splicing in human cells, we analyzed splicing of mRNAs encoding Bcl2 family apoptosis factors in a genome-wide siRNA screen. The screen identified many regulators of Bcl-x and Mcl1 splicing, notably an extensive network of cell-cycle factors linked to aurora kinase A. Drugs or siRNAs that induce mitotic arrest promote proapoptotic splicing of Bcl-x, Mcl1, and caspase-9 and alter splicing of other apoptotic transcripts. This response precedes mitotic arrest, indicating coordinated upregulation of prodeath splice variants that promotes apoptosis in arrested cells. These shifts correspond to posttranslational turnover of splicing regulator ASF/SF2, which directly binds and regulates these target mRNAs and globally regulates apoptosis. Broadly, our results reveal an alternative splicing network linking cell-cycle control to apoptosis.

Developmental consequences of alternative Bcl‐x splicing during preimplantation embryo development

Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl-x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl-x splicing, favoring production of Bcl-xS, an activator of cell death. Expression of Bcl-xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl-xL/Bcl-xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl-xS, with concomitant downregulation of Bcl-xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl-x splicing in favor of Bcl-xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl-x isoform expression, contributing to compromised preimplantation embryo development.

The Oncogenic Kinase Bcr-Abl Directly Regulates Splicing of BclX through a Quaternary Complex Coordinated by Nck-Beta and Sam-68 Adapter Proteins.

Abstract 2171Poster Board II-148Chronic Myelogenous Leukemia is the prototype of myeloproliferative disorder characterized by a reciprocal chromosomal translocation, involving the chromosomes 9 and 22 —t(9;22)-. The molecular consequence of this translocation is the generation of the Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cellular proliferation, alteration in cell-cell and cell-matrix adhesion, resistance to apoptosis which altogether are leukemogenesis landmarks.Although it is well established that Bcr-Abl-expressing leukemic cells are highly resistant to apoptotic cell death induced by chemotherapeutic drugs and a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway/s triggered by this oncogene has not been fully understood.The numerous experimental evidences collected in the last years highlight the crucial role played by alternative splicing in the control of apoptosis. Several pre-mRNAs for cell death factors are alternatively spliced, yielding isoforms with opposing functions during programmed cell death. A clear example is Bcl-x transcript which is alternatively spliced to produce the antiapoptotic Bcl-x(L) or the proapoptotic Bcl-x(S). Identical features are shared by another member of the Bcl-2 family, the myeloid cell leukemia-1 (MCL-1) gene which encodes two alternative splicing variants MCL-1S and MCL-1L displaying pro- and anti-apoptotic effects, respectively.CML-derived cell lines overexpress the antiapoptotic protein Bcl-x(L) and their erythroid differentiation is inhibited by Bcl-x(L).The data so far collected indicate that there is an extensive cross-talk among BCL-2 family members by virtue of their protein-protein interactions and the ratio of pro-apoptotic to anti-apoptotic proteins has been shown to be a major detereminant of the cell propensity to undergo apoptosis.Furthermore, it is well established that accelerated and blastic phases of the disease are characterized by deregulated WT1 expression. WT1/KTS- gene encodes for a transcription factor but the WT1/KTS+ isoform has been reported to localize into nuclear speckles, the major sub-nuclear structures enriched pre-messenger RNA and splicing factors.Based on the above premise we started investigating the possibility of an active involvement of Bcr-Abl as candidate regulator of splicing events affecting Bcl-x pre-mRNA.By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl in splicing assembling machinery. The data presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl, the RNA binding protein Sam68, the spliceosome ribonucleprotein hnRNPA1 and WT1. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta, Sam68, and hnRNPA1 supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68/hnRNPA1/WT1. Biochemical analysis carried-out by Pull-Down experiments has been further corroborated by immunofluorescence staining. RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/hnRNPA1/Bcr-Abl/WT1 might modulates splicing process of Bcl-x gene, whose function has been recently described as crucial in myeloproliferative disorders. Astoningshly, the data collected so far indicates that other mRNAs are pulled-down together with the quaternary complex. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam-68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex pre-mRNA splicing scenario. Disclosures:No relevant conflicts of interest to declare.

Heterogeneous Nuclear Ribonucleoprotein K Represses the Production of Pro-apoptotic Bcl-xS Splice Isoform

The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-x(L) and the pro-apoptotic Bcl-x(S) isoforms. By performing deletion mutagenesis on a human Bcl-x minigene, we have identified a novel exonic element that controls the use of the 5' splice site of Bcl-x(S). The proximal portion of this element acts as a repressor and is located downstream of an enhancer. Further mutational analysis provided a detailed topological map of the regulatory activities revealing a sharp transition between enhancer and repressor sequences. Portions of the enhancer can function when transplanted in another alternative splicing unit. Chromatography and immunoprecipitation assays indicate that the silencer element interacts with heterogeneous ribonucleoprotein particle (hnRNP) K, consistent with the presence of putative high affinity sites for this protein. Finally, down-regulation of hnRNP K by RNA interference enhanced splicing to Bcl-x(S), an effect seen only when the sequences bound by hnRNP K are present. Our results therefore document a clear role for hnRNP K in preventing the production of the pro-apoptotic Bcl-x(S) splice isoform.

Anticancer drugs affect the alternative splicing of Bcl-x and other human apoptotic genes

Inducing an apoptotic response is the goal of most current chemotherapeutic interventions against cancer. However, little is known about the effect of chemotherapeutic agents on the alternative splicing of apoptotic genes. Here, we have tested 20 of the mainstream anticancer drugs for their ability to influence the production of Bcl-x splice isoforms. We find that many drugs shift splicing toward the proapoptotic Bcl-x(S) splice variant in 293 cells. The drugs modulate splicing decisions most likely through signaling events because the splicing switch is not compromised by inhibiting de novo protein synthesis or the activity of caspases. Several drugs also shift Bcl-x splicing in cancer cell lines (MCF-7, HeLa, PC-3, PA-1, and SKOV-3), but the set of active drugs varies between cell lines. We also examined the effect of anticancer agents on the alternative splicing of 95 other human apoptotic genes in different cell lines. Almost every drug can alter a subset of alternative splicing events in each cell line. Although drugs of the same class often influence the alternative splicing of the same units in individual cell lines, these units differ considerably between cell lines, indicating cell line-specific differences in the pathways that control splicing.

Protein phosphatase 1 activation and alternative splicing of Bcl‐X and Mcl‐1 by EGCG + ibuprofen

Epigallocatechin-3-gallate (EGCG) and ibuprofen synergistically act to suppress proliferation and enhance apoptosis of prostate cancer cell lines, PC-3 and LNCaP. The purpose of this study was to investigate the mechanism of underlying this synergism. Most interestingly, EGCG + ibuprofen treatment in PC-3 cells resulted in altering the ratio of the splice variants of Bcl-X and Mcl-1, downregulating the mRNA levels of anti-apoptotic Bcl-X(L) and Mcl-1(L) with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-X(s) and Mcl-1(s). However, there were no apparent changes in splicing variants in either ibuprofen or EGCG treated cells. Induction of alternative splicing was correlated with increased activity of protein phosphatase 1 (PP1) in EGCG + ibuprofen-treated cells, since pretreatment with calyculin A and tautomycin blocked EGCG + ibuprofen-induced alternative splicing in PC-3 cells in contrast to pretreatment with okadaic acid. On the other hand, EGCG + ibuprofen treatment in LNCaP cells did not alter splicing variants of Bcl-X and Mcl-1, despite the increase in protein phosphatase activity. In both cell lines, EGCG + ibuprofen inhibited cell proliferation synergistically. Taken together, this study demonstrate for the first time that EGCG + ibuprofen upregulated PP1 activity, which in turn induced alternative splicing of Bcl-X and Mcl-1 in a cell-type specific manner. Our study also demonstrates that the activation of PP1 contributes to the alternative splicing of Mcl-1.

Emetine Regulates the Alternative Splicing of Bcl-x through a Protein Phosphatase 1-Dependent Mechanism

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.