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...

FR901464 is a potent antitumor natural product that binds to the splicing factor 3b complex and inhibits premRNA splicing. Its analogue, meayamycin, is two orders of magnitude more potent as an antiproliferative agent against human breast cancer MCF-7 cells. Here, we report the picomolar antiproliferative activity of meayamycin against various cancer cell lines and multidrug-resistant cells. Time-dependence studies implied that meayamycin may form a covalent bond with its target protein(s). Meayamycin inhibited pre-mRNA splicing in HEK-293 cells but not alternative splicing in a neuronal system. Meayamycin exhibited specificity toward human lung cancer cells compared with nontumorigenic human lung fibroblasts and retained picomolar growth-inhibitory activity against multidrug-resistant cells. These data suggest that meayamycin is a useful chemical probe to study premRNA splicing in live cells and is a promising lead as an anticancer agent.