Anticancer drugs affect the alternative splicing of <i>Bcl-x</i> and other human apoptotic genes

Shkreta, Lulzim; Froehlich, Ulrike; Paquet, Éric R.; Toutant, Johanne; Elela, Sherif Abou; Chabot, Benoît

doi:10.1158/1535-7163.mct-08-0192

Cited by 61 publications

(64 citation statements)

References 44 publications

(33 reference statements)

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“…It is noted, however, that both affect calcium physiology. Several anticancer drugs have been recently shown to affect alternative splicing of a group of apoptosis-related test genes (52). However, the drug treatments were for 24 h, and thus, the effects on splicing could be an adaptive response rather than a direct effect.…”

Section: Discussionmentioning

confidence: 99%

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing

Younis

Berg

Kaida

et al. 2010

Molecular and Cellular Biology

111

125

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Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.Extensive posttranscriptional processing of eukaryotic premRNA is required for the biogenesis of mRNAs, including 7-methylguanosine cap addition at the 5Ј end, cleavage and polyadenylation at the 3Ј end, and splicing of introns. The vast majority of genes in complex eukaryotes contain multiple introns that need to be spliced out by the spliceosome with high fidelity in order to generate the open reading frame (ORF) and the 5Ј and 3Ј untranslated regions (UTR) carried by the exons. Splicing depends on the recognition of short consensus sequences at the intron-exon boundaries and within introns by a set of small nuclear ribonucleoprotein (snRNP) complexes (consisting of snRNPs U1, U2, U4, U5, U6, U11, U12, U4atac, and U6atac) and a large number of proteins, including spliceosomal proteins and positively as well as negatively acting splicing modulators (12, 59). Serine-arginine-rich (SR)-domain-containing proteins (36) generally serve to promote constitutive splicing. They also modulate alternative splicing by binding to intronic or exonic splicing enhancer (ISE or ESE, respectively) sequences (5, 20). Other pre-mRNA binding proteins, such as hnRNPs, that lack SR domains regulate splicing by binding to intronic or exonic splicing suppressor (ISS or ESS, respectively) sites and also act as general splicing modulators (14, 62).Alternative splicing allows for a single gene to express different isoforms of mRNA, thus playing a major role in contributing to the cellular complexity in higher eukaryotes without the need to expand the genome (6). Global surveying of the human transcriptome estimates that up to 95% of multiexon genes undergo alternative splicing (46, 60). Importantly, these events are highly regulated by numerous splicing factors in a tissue type-, developmental stage-, and signal-dependent manner. Aberrations in splicing due to...

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Section: Discussionmentioning

confidence: 99%

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing

Younis

Berg

Kaida

et al. 2010

Molecular and Cellular Biology

111

125

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“…To identify other genes similarly regulated by EJC components, we monitored 96 alternative splicing events (ASEs) in apoptotic and cancer-related genes using a high-throughput RT-PCR platform (30,64,(75)(76)(77). We looked for events that, like for Bcl-x, would be affected by depleting EJC components but not Upf1.…”

Section: Components More Prevalently Associated With Ejc Function Affmentioning

confidence: 99%

Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

Michelle

Cloutier

Toutant

et al. 2012

Molecular and Cellular Biology

Self Cite

120

142

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Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x S splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

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“…Most of chemotherapeutic drugs work by impairing mitosis and/or inducing apoptosis. Several anticancer drugs target DNA or enzyme acting on the DNA [1]. The resistance of tumor cells to different antineoplastic agent is an obstacle for cancer chemotherapy.…”

Section: Introductionmentioning

confidence: 99%

Influence OF anticancer drugs on DNA methylation in liver of female mice

Hanafy¹,

Salem²,

El-Aziz³

et al. 2011

AJMB

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Epigenetic changes such as DNA methylation regulate gene expression in normal development. Methotrexate and Adriamycine are antineoplastic drugs that target DNA and enzymes acting on DNA. We aimed to evaluate their cytotoxic effect on cell lines and on female mice to investigate the in vivo influence of both drugs on the DNA methylation and subsequently the protein expression. The total level of DNA methylation showed a significant reduction from 62.2% to 36.7%, 36.6% as compared to control group, when using different doses of MTX and ADR. Hepatic protein pattern revealed five bands with low MW (16 -6.1 KDa) in acute and LD50 doses. In conclusion DNA methylation is influenced by anticancer drugs in a dose-dependent manner. Some specific protein fragments may be considered as a potential markers associated with high dose of anticancer drugs.

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

Cited by 61 publications

References 44 publications

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing

Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

Influence OF anticancer drugs on DNA methylation in liver of female mice

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