A high-throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes.
J Biomol Screen
; 18(9): 1110-20, 2013 Oct.
Article
em En
| MEDLINE
| ID: mdl-23771823
ABSTRACT
The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Precursores de RNA
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Splicing de RNA
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Spliceossomos
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Bibliotecas de Moléculas Pequenas
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Ensaios de Triagem em Larga Escala
Limite:
Humans
Idioma:
En
Revista:
J Biomol Screen
Ano de publicação:
2013
Tipo de documento:
Article