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RNA. 2009 Jan;15(1):153-75. doi: 10.1261/rna.1332609. Epub 2008 Nov 24.

Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation.

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  • 1Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.

Abstract

The removal of intervening sequences from a primary RNA transcript is catalyzed by the spliceosome, a large complex consisting of five small nuclear (sn) RNAs and more than 150 proteins. At the start of the splicing cycle, the spliceosome assembles anew onto each pre-mRNA intron in an ordered process. Here, we show that several small-molecule inhibitors of protein acetylation/deacetylation block the splicing cycle: by testing a small number of bioactive compounds, we found that three small-molecule inhibitors of histone acetyltransferases (HATs), as well as three small-molecule inhibitors of histone deacetylases (HDACs), block pre-mRNA splicing in vitro. By purifying and characterizing the stalled spliceosomes, we found that the splicing cycle is blocked at distinct stages by different inhibitors: two inhibitors allow only the formation of A-like spliceosomes (as determined by the size of the stalled complexes and their snRNA composition), while the other compounds inhibit activation for catalysis after incorporation of all U snRNPs into the spliceosome. Mass-spectrometric analysis of affinity-purified stalled spliceosomes indicated that the intermediates differ in protein composition both from each other and from previously characterized native A and B splicing complexes. This suggests that the stalled complexes represent hitherto unobserved intermediates of spliceosome assembly.

PMID:
19029308
[PubMed - indexed for MEDLINE]
PMCID:
PMC2612777
Free PMC Article
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