Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation
Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation
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DOI：
10.1261/rna.1332609
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发表时间：
2009-01-01
期刊：
影响因子：
4.5
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Kuhn, Andreas N.;Van Santen, Maria A.;Luehrmann, Reinhard
剪接体催化从初级 RNA 转录物中去除干扰序列，剪接体是一个由 5 个小核 (sn) RNA 和 150 多种蛋白质组成的大型复合物。在剪接周期开始时，剪接体以有序的过程重新组装到每个前 mRNA 内含子上。在这里，我们展示了几种蛋白质乙酰化/脱乙酰化的小分子抑制剂阻断剪接周期：通过测试少量的生物活性化合物，我们发现三种组蛋白乙酰转移酶（HAT）的小分子抑制剂（HAT）以及三种组蛋白脱乙酰酶（HDAC）的小分子抑制剂在体外阻断前mRNA剪接。通过纯化和表征停滞的剪接体，我们发现剪接循环在不同阶段被不同的抑制剂阻断：两种抑制剂仅允许形成A样剪接体（由停滞复合物的大小及其snRNA组成决定），而其他化合物在所有U snRNP掺入剪接体后抑制催化激活。亲和纯化的停滞剪接体的质谱分析表明，中间体之间的蛋白质组成不同，并且与先前表征的天然 A 和 B 剪接复合物不同。这表明停滞的复合物代表了迄今为止未观察到的剪接体组装的中间体。
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.