Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.
Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.
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DOI：
10.1371/journal.pone.0048084
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kornblihtt AR
中科院分区：
文献类型：
--
作者：
Schor IE;Llères D;Risso GJ;Pawellek A;Ule J;Lamond AI;Kornblihtt AR
染色质结构是转录和mRNA加工之间功能偶联的一个重要因素，它不仅通过调控可变剪接事件，而且通过在组成性剪接过程中促进外显子识别来发挥作用。我们观察到，神经母细胞瘤细胞膜电位去极化会引发普遍的组蛋白乙酰化并调控可变剪接，导致SR蛋白在核斑点中聚集。这促使我们分析染色质结构对剪接因子分布和动态的影响。在此，我们表明组蛋白过度乙酰化的诱导导致多种剪接因子在不同细胞类型的斑点中积累。此外，在与抑制性染色质相关的异染色质蛋白HP1α耗竭后也观察到了类似的效应。我们使用先进的成像方法详细分析了斑点区室的结构组织和剪接因子的核分布，以及研究了剪接因子之间的直接相互作用及其在体内与染色质的关联。结果支持这样一种模型：正常染色质结构的扰动会降低剪接因子对新生RNA的招募效率，从而导致它们在斑点中积累，而斑点会缓冲核质中游离分子的数量。为了验证这一点，我们通过iCLIP技术分析了通用剪接因子U2AF65对新生RNA的招募，以此作为监测早期剪接体组装的一种方式。我们证明，组蛋白过度乙酰化确实会降低U2AF65对大量3′剪接位点的招募，这与其定位的变化一致。此外，在斑点中达到最大积累之前，约20%的基因已经显示出结合减少的趋势，而U2AF65似乎增加了其与位于斑点中的非编码RNA MALAT1的结合。总之，综合的成像和生化方法支持这样一种模型：染色质结构对于通用和调节性剪接因子高效共转录招募到前体mRNA是必不可少的。
Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA.
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DOI：
10.1083/jcb.201004030
发表时间：
2010-10-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Huranová M;Ivani I;Benda A;Poser I;Brody Y;Hof M;Shav-Tal Y;Neugebauer KM;Stanek D
通讯作者：
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发表时间：
2009-02-23
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影响因子：
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作者：
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通讯作者：
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影响因子：
16.8
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影响因子：
16
作者：
Görnemann, J;Kotovic, KM;Neugebauer, KM
通讯作者：
Neugebauer, KM
影响因子：
16.8
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Muchardt, C