Identification of small molecule inhibitors of pre-mRNA splicing.

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.

Exo70, a subunit of the exocyst complex, interacts with SNEV(hPrp19/hPso4) and is involved in pre-mRNA splicing.

The Cdc5L (cell division cycle 5-like) complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPrp19, also known as SNEV or hPso4, which is involved in cellular life-span regulation and proteasomal breakdown. In a recent large-scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell-cycle regulation, DNA repair, the ubiquitin-proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including four members of the exocyst complex, were identified. In the present paper we report that Exo70 interacts directly with SNEV(hPrp19/hPso4) and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pre-mRNA splicing in vitro. Furthermore, Exo70 influences the splicing of a model substrate as well as of its own pre-mRNA in vivo. In addition, we found that Exo70 is alternatively spliced in a cell-type- and cell-age- dependent way. These results suggest a novel and unexpected role of Exo70 in nuclear mRNA splicing, where it might signal membrane events to the splicing apparatus.

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP.

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.

Large-scale proteomic analysis of the human spliceosome.

In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.

A novel function for human factor C1 (HCF-1), a host protein required for herpes simplex virus infection, in pre-mRNA splicing.

Human factor C1 (HCF-1) is needed for the expression of herpes simplex virus 1 (HSV-1) immediate-early genes in infected mammalian cells. Here, we provide evidence that HCF-1 is required for spliceosome assembly and splicing in mammalian nuclear extracts. HCF-1 interacts with complexes containing splicing snRNPs in uninfected mammalian cells and is a stable component of the spliceosome complex. We show that a missense mutation in HCF-1 in the BHK21 hamster cell line tsBN67, at the non-permissive temperature, inhibits the protein's interaction with U1 and U5 splicing snRNPs, causes inefficient spliceosome assembly and inhibits splicing. Transient expression of wild-type HCF-1 in tsBN67 cells restores splicing at the non-permissive temperature. The inhibition of splicing in tsBN67 cells correlates with the temperature-sensitive cell cycle arrest phenotype, suggesting that HCF-1-dependent splicing events may be required for cell cycle progression.