The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation.
The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation.
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DOI：
10.1371/journal.pgen.1000595
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发表时间：
2009-08
期刊：
影响因子：
4.5
通讯作者：
Grabowski PJ
中科院分区：
文献类型：
--
作者：
Dembowski JA;Grabowski PJ
替代的前mRNA剪接通过从单个初级转录本产生相关的mRNAs来调节基因组的转录输出，从而扩大蛋白质多样性。一个基本的悬而未决的问题是，尽管信息贫乏的序列基序被识别，剪接因子如何在靶底物的选择中实现特异性。CUGBP2剪接调节因子在NMDAR1受体NI外显子的脑区特异性沉默中起关键作用。然而，该因子用于特定目标外显子选择的序列基序及其在剪接沉默中的作用尚不清楚。在这里，我们使用化学修饰足迹法将CUGBP2的接触位置映射到紧密位于NI外显子分支位置边界的富Gu基序，并证明了这种特定的基序排列在调节分支点形成方面的机械作用。鉴定了一组新的靶标外显子，这些外显子具有相似的基序结构，并被CUGBP2沉默，表明对分支位点-周长结合模型的普遍支持。这些结果揭示了CUGBP2的自我调节作用，这表明它与CUGBP2转录本第6外显子上游分支位点周围的功能显著的RNA基序直接相互作用。周长结合模型解释了CUGBP2如何有效地拥抱分支位点区域，以实现选择外显子靶标和微调替代剪接模式所需的特异性。选择性剪接是一个精确控制的过程，它决定了成熟的mRNA转录本中是否包含或跳过外显子。控制选择性剪接的因子与外显子或侧翼内含子中的RNA序列基序结合，并指导组织和发育的特异性剪接事件。CUGBP2是选择性剪接的双重功能调节器，根据其结合基序的上下文，可以导致目标外显子的包含或跳过。以前，该蛋白的调节机制及其靶基序的位置意义尚未确定。在本研究中，作者剖析了CUGBP2跳过外显子的机制，并证明了功能参考点周围的特定基序配置与这一事件密切相关。此外，这种调控机制被证明具有普遍意义，因为新的CUGBP2靶标外显子包含类似的基序排列。这一组中最有趣的是CUGBP2转录本本身的一个外显子。这项研究强调了功能参照点在另一种剪接因子调控的特异性中的重要性，并揭示了CUGBP2的一种新的自我调节作用。
Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter–binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns. Alternative splicing is a precisely controlled process that determines whether an exon will be included or skipped in the mature mRNA transcript. Factors that control alternative splicing bind to RNA sequence motifs in the exon or flanking introns and guide tissue and developmental specific splicing events. CUGBP2 is a dual functional regulator of alternative splicing that can cause inclusion or skipping of a target exon, depending on the context of its binding motifs. Previously, the mechanisms of regulation by this protein and the positional significance of its target motifs have not been characterized. In this study, the authors dissected the mechanism of exon skipping by CUGBP2 and demonstrate that a specific configuration of motifs at the perimeters of a functional reference point are intimately involved in this event. Furthermore, this mechanism of regulation is shown to have general significance because novel CUGBP2 target exons contain a similar arrangement of motifs. The most interesting of this group is an exon within the CUGBP2 transcript itself. This study underscores the importance of a functional reference point in the specificity of regulation by an alternative splicing factor and reveals a novel autoregulatory role for CUGBP2.
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