期刊
RNA
卷 15, 期 1, 页码 153-175出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.1332609
关键词
pre-mRNA splicing; spliceosome; proteomics; histone deacetylase/HDAC; histone acetyltransferase/HAT
资金
- DAAD
- Deutsche Forschungsgemeinschaft
- EU [518238]
- Volkswagenstiftung
- HFSP [RGY0056/2004]
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.
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