The Biflavonoid Isoginkgetin Is a General Inhibitor of Pre-mRNA Splicing

Abstract

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. 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) 2The abbreviations used are: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; FRT, flp recombinase target; RT, reverse transcription; AdML, adenovirus major late; TPI, triose-phosphate isomerase. 2The abbreviations used are: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; FRT, flp recombinase target; RT, reverse transcription; AdML, adenovirus major late; TPI, triose-phosphate isomerase. and more than 100 polypeptides (reviewed in Ref. 1Matlin A.J. Moore M.J. Adv. Exp. Med. Biol. 2007; 623: 14-35Crossref PubMed Scopus (68) Google Scholar). Within the spliceosome, intron excision occurs in two chemical steps: 1) 5′ splice site cleavage accompanied by lariat formation at the branch point adenosine and 2) 3′ 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