Abstract
Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed "madrasin," i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells.
Highlights
There is a need for new small molecule pre-mRNA splicing inhibitors as biotools
DDD00107587 Alters Pre-mRNA Splicing in Vivo—we investigated the effect of DDD00107587 on the splicing of endogenous pre-mRNAs in vivo
Loss of Cajal Bodies after Treatment with DDD00107587— we investigated the effect of DDD00107587 on subnuclear structures and on the organization of pre-mRNA splicing factors within the nucleus
Summary
There is a need for new small molecule pre-mRNA splicing inhibitors as biotools. Results: High throughput screening resulted in the identification of small molecule splicing inhibitors that are active in vitro and in cells. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. The high throughput in vitro splicing assay developed by Samatov et al [17] allows the identification of small molecule splicing inhibitors that affect either spliceosome assembly, activation, and/or step 1 catalysis This is achieved by using the PM5 pre-mRNA, which lacks both the 3Ј-splice site and 3Ј exon of a typical pre-mRNA and stops the splicing reaction after C complex formation. We characterize the effect of one of these compounds, DDD00107587 (madrasin), both on splicing in vitro and in cells
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