A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators

Abstract

Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a twocolor fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.digoxin ͉ fluorescent reporter ͉ plant steroids ͉ MAPT ͉ FTDP-17 D isruption of exon recognition and misregulation of alternative splicing are a common cause of human disease. Conditions linked to errors in pre-mRNA splicing include autoimmune disorders, neurodegenerative diseases, cystic fibrosis, growth hormone deficiency, muscular dystrophy, and cancer progression (1-3). Thus, the RNA splicing machinery is an important potential target for drug development. However, few drugs have been identified that specifically target the splicing reaction, and the impact of existing drugs on splicing regulation is not routinely examined.One disease stemming from misregulation of alternative splicing is frontotemporal dementia with parkinsonism on chromosome 17 (FTDP-17) (4). FTDP-17 is caused by mutations affecting exon 10 of the microtubule-associated protein tau (MAPT) gene that increase inclusion of exon 10 in the mRNA. Exon 10 encodes the fourth microtubule-binding domain of the protein, and elevated levels of the four-domain form of tau evidently lead to neurofibrillary tangles and subsequent neurodegeneration. Thus, there is significant therapeutic interest in modulating the splicing of this exon. Multiple factors are known to affect MAPT exon 10 splicing (4-9). This combinatorial control is typical of alternative splicing patterns and points to the difficulties in developing splicingtargeted therapeutics. Interfering with individual factors often only partially affects a given splicing pattern, and each factor can affect many unrelated exons in addition to the therapeutic target.Finding compounds that modulate alternative splicing requires effective high-throug...

Systematic evaluation of the effect of common SNPs on pre-mRNA splicing

“…Third, the genomic region had to be inserted while preserving the ORF of GFP. The site-directed mutagenesis experiments and data obtained from FACS-based (Figure 3.12) and immunoblot analyses (Figure 3.13) confirmed the efficiency of the current construct (RFP-PGM2L1-pEGFP-N1; reporters have poor dynamic range or do not distinguish compounds affecting splicing from those altering transcription or translation (Stoilov et al, 2008). Indeed, three recent studies demonstrate the utility of dual-color reporter systems in improving the dynamic range and discriminating changes in alternative splicing from changes in transcription or translation.…”

“…Two of these systems (Newman et al, 2006;Orengo et al, 2006) may require modification of a test exon to adapt it to the reporter, which may change its regulatory properties (Stoilov et al, 2008). The last evolved system argued for its flexibility to accommodate a variety of test exons from different genes in a high-throughput trend (Stoilov et al, 2008).…”

The anti-tumor drug E7107 reveals an essential role for SF3b in remodeling U2 snRNP to expose the branch point-binding region

“…The identification of specific roles for SF3b via the use of E7 and spliceostatin A, as well as other recent work using small molecules to examine splicing, underscores the utility of these compounds for elucidating specific functions of the highly complex, dynamic factors in the spliceosome (Pilch et al 2001;Muraki et al 2004;Kaida et al 2007;Kotake et al2007;O'Brien et al 2008;Stoilov et al 2008;Sumanasekera et al 2008;Kuhn et al2009;Younis et al 2010). Although the binding affinity of SF3b to E7 is highly correlated with its cell growth inhibition activity (Kotake et al 2007), the mechanistic basis for the anti-tumor effect of E7 is not known.…”

Splicing inhibition of U2AF 65 leads to alternative exon skipping

“…As the second approach to identifying RNA sequence requirements in SMN pre-mRNA for splicing inhibition, we used a previously reported construct that includes upstream β-globin exon 1 and downstream GFP/RFP exons as a reporter for the splicing of middle test exon (38). We inserted SMN exon 7 and its upstream 573-nt intron and downstream 424-nt intron between the β-globin and GFP/RFP exons (SMN1/2-E7; Fig.…”