In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms - thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.The removal of introns from nascent transcripts by the process of pre-mRNA (precursor to messenger RNA) splicing is an essential step in eukaryotic gene expression. Splicing is mediated by the spliceosome, a highly dynamic, multimegadalton machine composed of five small stable nuclear RNAs (snRNAs) 2 and more than 100 polypeptides (reviewed in Ref. 1). Within the spliceosome, intron excision occurs in two chemical steps:1) 5Ј splice site cleavage accompanied by lariat formation at the branch point adenosine and 2) 3Ј splice site cleavage accompanied by exon ligation. Both of these steps are readily observable in in vitro reactions containing crude nuclear extract and ATP as an energy source. In such reactions, spliceosome assembly occurs in a distinctly stepwise fashion. First, the pre-mRNA substrate is coated with a heterogeneous mixture of RNA-binding proteins (referred to as H complex). Interaction of U1 snRNP (U1 snRNA and its associated proteins) with the 5Ј splice site and recognition of the branch point adenosine by U2 snRNP generates an early commitment complex (E or CC complex). A subsequent ATP-dependent step stabilizes the U2 snRNP-branch point interaction, resulting in formation of the prespliceosome (A complex). Entry of the U4/U5/U6 tri-snRNP to form B complex is followed by multiple structural rearrangements, which produce the catalytically active C complex, wherein the two chemical steps of splicing occur. Finally, the ligated exon and lariat products are released, and the remaining spliceosome components are disassembled.In the more than two decades since its initial description (2, 3), a wealth of information has been gleaned regarding the parts list of the spliceosome, its gross assembly/disassembly pathway, certain key local structural int...

The ␣-actinin gene has a pair of alternatively spliced exons. The smooth muscle (SM) exon is repressed in most cell types by polypyrimidine tract binding protein (PTB). CELF (CUG-BP and ETR3-like factors) family proteins, splicing regulators whose activities are altered in myotonic dystrophy, were found to coordinately regulate selection of the two ␣-actinin exons. CUG-BP and ETR3 activated the SM exon, and along with CELF4 they were also able to repress splicing of the NM (nonmuscle) exon both in vivo and in vitro. Activation of SM exon splicing was associated with displacement of PTB from the polypyrimidine tract by binding of CUG-BP at adjacent sites. Our data provides direct evidence for the activity of CELF proteins as both activators and repressors of splicing within a single-model system of alternative splicing, and suggests a model whereby ␣-actinin alternative splicing is regulated by synergistic and antagonistic interactions between members of the CELF and PTB families.

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.