Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation.
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.
Kuhn
Andreas N
AN
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
van Santen
Maria A
MA
Schwienhorst
Andreas
A
Urlaub
Henning
H
Lührmann
Reinhard
R
D000107:Acetylation; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000187:drug effects / Q000378:metabolism*
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D012322:RNA Precursors; D017413:Ribonucleoprotein, U2 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C552126:U11-U12 small nuclear ribonucleoprotein; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases