In order to identify the alternative splicing events controlled by specific splicing regulators, Blanchette et al. 17 made use of a recent experimental innovation - the combination of the silencing of regulators with splicing-sensitive microarrays to detect the effects. This technology has the potential not only rapidly to identify the targets of these regulators throughout the genome, but also to tease apart their combinatorial control. Blanchette et al. 17 investigated four well-characterized and highly expressed splicing regulators - the SR proteins dASF (Drosophila alternative splicing factor, the homolog of human ASF, also known as splicing factor 2; SF2) and B52 (the Drosophila equivalent of the mammalian SRp55 protein) and the hnRNPs PSI and hrp48. They developed a splicing-sensitive microarray platform to monitor around 3,000 annotated alternatively spliced genes in the Drosophila genome. As in other studies 21519, oligonucleotide probes were designed to span annotated alternatively spliced junctions, with control probes across the relevant constitutive junctions (that is, junctions that are always spliced together) and within exons. In order to silence the regulators by RNA interference (RNAi), Drosophila SL2 cells were treated with double-stranded RNAs specific for each regulator gene. As these regulators are likely to be associated not only with splicing but also with other RNA-processing functions, Blanchette et al. 17 initially feared that a decrease in the levels of the regulators might affect pre-mRNA processing in such a general manner that the array data would be uninterpretable. Fortunately, hierarchical clustering of multiple RNAi experiments showed characteristic and reproducible splicing responses.

The RNA isolated from the siRNA-treated cells was then hybridized on the microarrays. Each regulator affected distinct sets of splice junctions, but the results revealed significant overlap in the targets of dASF/SF2 and B52/SRp55, consistent with the observation that SR proteins can complement one another on particular targets (see references cited in 17). The authors also observed that almost all the events affected by PSI are also controlled by hrp48, suggesting that hrp48 is a required partner for PSI. Interestingly, this relationship does not seem to be mutual, but further experiments are necessary to confirm this finding. Figure 1 illustrates this scenario: regulator II is an obligatory partner to I, but I is not required for II.

Consistent with the notion that dASF/SF2 is a general regulator of alternative splicing, its knockdown affected the largest number of events (319 events). Conversely, PSI, a more specific regulator of alternative splicing, affected the fewest events (43 events). In order to obtain evidence for direct binding of B52/SRp55, a positional weight matrix of an identified binding site for B52/SRp55 was used to scan the exon-intron boundaries of splicing events affected by knockdown of the factor. The motif was indeed specifically over-represented at the 5' splice sites of exons at which splicing is reduced when B52/SRp55 is knocked down. Together, the findings revealed a network of tens to hundreds of alternative splicing events that are regulated by individual or combinations of splicing regulators.

As acknowledged by Blanchette et al. 17, questions still remain about why some targets are affected similarly by different regulators. There might be co-occurring binding sites in the same set of exons, or the regulators might have overlapping binding specificities, or the regulators might be interacting with another common regulator already present. Knockdowns and overexpression of more splicing factors, and parallel analyses of the sequence similarity of regulated exons (and flanking introns), as well as the RNA binding domain characteristics of the splicing regulators, should clarify these questions.

Various ways of correcting splicing defects have been sought. Disease-associated exons can be induced to skip by antisense oligonucleotides 2021, or exon inclusion can be restored by synthetic exon-specific effectors 22 or mediated by RNA trans-splicing 23. Although they are effective in correcting the splicing defect, these methods are not readily adaptable to high-throughput analysis with a view to finding drugs that rescue aberrant splicing events.

In another attempt to correct splicing defects, Sorel et al. 18 investigated the inhibition of the recombinant SR protein ASF/SF2 by small-molecule compounds. This approach lends itself well to high-throughput methods. In earlier work the authors had found that drugs interfering with the kinase activity of topoisomerase I (topo I) affect the phosphorylation status of SR proteins and prevent spliceosomal assembly (see reference cited in 18). Building on this, they screened 2,500 chemical compounds for the inhibition of topo I phosphorylation of ASF/SF2, and obtained 28 potent inhibitors (Figure 2a). To determine whether these compounds selectively inhibit in vitro splicing of pre-mRNAs, the authors tested these 28 compounds on two pre-mRNA substrates: a synthetic single-intron pre-mRNA derived from the adenovirus major late-transcription unit (Minx), and a derivative of the human β-globin gene that contains three exonic splicing enhancers resembling a high-affinity ASF/SF2-binding site (βglo-3S). Three compounds had strong inhibitory effects on βglo-3S, whose splicing depends on ESE sequences, but only one of the three affected Minx pre-mRNA splicing, which is independent of ESE sequences (Figure 2a).

To determine which stage of spliceosome assembly was affected, Soret et al. 18 then added ³²P-labeled βglo-3S pre-mRNA to HeLa nuclear extracts and incubated the mixture with each of the three compounds. An early step of assembly must have been disrupted, as no splicing complexes were formed. Realizing that monitoring spliceosome assembly would be a more straightforward way of identifying compounds that affect splicing, the authors screened 1,500 small molecules and identified 25 candidates (Figure 2b). Surprisingly, these all have similar structures, being indole derivatives of the pyridocarbazole, benzopyridoindole or pyrido-pyrrolo-isoquinoline classes.

The next question was whether these compounds were targeting ASF/SF2 directly, or the kinase activity of topo I kinase, or both. Taking advantage of the strong intrinsic fluorescence of one of the compounds, the authors found that it interacted with ASF/SF2 directly rather than with topo I (80% of drug fluorescence was quenched upon binding to ASF/SF2). They found that while the overall structure of ASF/SF2 is important, the RS domain of the protein is the major drug-binding element. The RS domain contains the repeated arginine (R)-serine (S) dipeptides that characterize SR proteins. These domains are present not only in the SR proteins but also in canonical splicing factors such as the U1-snRNP-specific proteins U1-70K and U2AF. Their inhibition could have effects on general RNA splicing and this emphasizes the importance of identifying all possible targets of potential drugs to avoid problems of toxicity. Figure 3 illustrates three possible classes of drug, from the most potentially toxic to the most specific and safest.

In a subsequent experiment, Soret et al. 18 screened more than 200 indole derivatives for their ability to inhibit the splicing of βglo-3S by ASF/SF2 or the splicing of βglo-SRp55 by SRp55 (Figure 2c). In the latter construct, the sequences with high affinity for ASF/SF2 have been replaced by the optimal binding site for SRp55. Satisfyingly, they discovered that some drugs are specific for one of the SR proteins while some affected both. In a further experiment, they created a gain-of-function mutation in HeLa cells, in which a G-to-A change in an intron of the E1α pyruvate dehydrogenase (PDH) gene generates an ESE that binds the SR protein SC35, and activates a cryptic 5' splice site downstream of the mutation in the same intron. Consistent with indole derivatives selectively inhibiting splicing depending on the SR proteins involved, two drugs were shown to inhibit the use of the cryptic splice site, presumably by affecting the binding of SC35 (Figure 2d). The type of mutation created in PDH is likely to be similar to splicing mutations that generate defective proteins in humans, and provides an avenue for therapeutic intervention (Figure 4).

As icing on the cake, Soret et al. 18 looked at the effects of their indole derivatives on the splicing of the pre-mRNA of the human immunodeficiency virus HIV-1. This pre-mRNA is regulated by alternative splicing involving SR proteins such as ASF/SF2 and SC35. Chronically infected human promonocytic U1 cell lines, which can be stimulated to produce large quantities of HIV-1 pre-mRNA, were treated with a number of indole derivatives 18. Several of these were shown specifically to affect HIV-1 alternative splicing, and abolished HIV-1 virion production. Neither cell viability nor the splicing profiles of endogenous genes were affected, indicating the potentially low toxicity of this treatment. This remarkable discovery opens new approaches to treatment of HIV-1 infection by indole derivatives via the interruption of ESE-mediated alternative splicing.

The studies of Blanchette et al. 17 and Soret et al. 18 are the first large-scale screens for the genome-wide targets of a small set of splicing regulators and for compounds that disrupt a specific class of splicing regulators. One can envisage combining the power of the two methods. For example, splicing-specific microarrays can query alternative splicing events affected by particular compounds. Comparing these events to alternative events affected by knockdowns of particular regulators may identify the candidate regulators affected by the compounds. More comprehensive siRNA screens of other RNA-binding proteins 24 using splicing-specific microarrays could point to new roles for splicing modulation. Looking forward, a large-scale mapping of regulators to targets in humans, and of compounds to regulators, combined with computational extraction of potential cis-regulatory binding sites, will be essential in the screening and correction of splicing defects in human disease.