Exon silencing by UAGG motifs in response to neuronal excitation.
Exon silencing by UAGG motifs in response to neuronal excitation.
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DOI：
10.1371/journal.pbio.0050036
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发表时间：
2007-02
期刊：
影响因子：
9.8
通讯作者：
Grabowski PJ
中科院分区：
文献类型：
--
作者：
An P;Grabowski PJ
选择性前体mRNA剪接通过在与突触的通信和连接相关的蛋白质中产生功能多样性而在神经元中发挥重要作用。NMDA R1受体的CI盒是响应于细胞激发而显示外显子跳跃增加的多种外显子之一，但这种剪接响应性的分子性质尚未被理解。在这里，我们调查的诱导变化的剪接的CI盒外显子在原代大鼠皮质培养物中的KCl诱导的去极化使用表达测定与紧密的神经元特异性读出的分子基础。在该系统中，响应于神经元兴奋的外显子沉默由多个UAGG型沉默基序介导，并且将基序转移到组成性外显子通过获得功能赋予类似的响应性。从处理和模拟处理的皮质培养物中制备的提取物中的蛋白质结合UAGG基序的生化分析显示，与激发平行的核hnRNP A1-RNA结合活性的增加。NMDA受体和钙信号传导在诱导的剪接反应中的作用的证据通过使用特异性拮抗剂以及信号传导途径的细胞渗透性抑制剂来显示。最后，一个更广泛的作用，外显子跳跃反应涉及额外的外显子与UAGG相关的沉默基序，和转录参与突触功能。这些结果表明，在转录后水平上，可兴奋的外显子，如CI盒可能参与神经元对过度刺激的适应性反应的策略。NMDA R1受体外显子21（CI盒）的外显子跳跃是由UAGG型沉默基序介导的，提出了一种机制，外部刺激通过该机制与神经元中的核剪接机制进行通信。蛋白质结构的模块特征在转录后通过选择性前mRNA剪接过程进行调节。刺激或应激神经元的条件可以诱导某些剪接模式的变化，这表明细胞通路可以利用剪接的灵活性来调节其蛋白质活性以适应或生存。虽然诱导型剪接开关（或诱导型外显子）的现象已经有了很好的记录，但这些奇怪变化的分子基础仍然是神秘的。我们描述的方法来研究如何谷氨酸NMDA受体，这是一个基本组成部分的神经元间的信号和可塑性，经历了一个诱导开关在其剪接模式在初级神经元。这种剪接开关促进编码Cl盒蛋白模块的外显子的跳跃，其被认为在神经元活动期间将信号从膜传递到细胞核。我们表明，这种诱导的剪接事件在神经元中由外显子沉默的三部分（UAGG型）序列代码调节，并证明了外显子跳跃反应在具有已知突触功能的转录本中具有更广泛的作用，这些转录本也具有类似的序列代码。
Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation. Exon skipping of NMDA R1 receptor exon 21 (CI cassette) is mediated by UAGG-type silencing motifs, presenting a mechanism by which external stimuli communicate to the nuclear splicing machinery in neurons. The modular features of a protein's architecture are regulated after transcription by the process of alternative pre-mRNA splicing. Conditions that excite or stress neurons can induce changes in some splicing patterns, suggesting that cellular pathways can take advantage of the flexibility of splicing to tune their protein activities for adaptation or survival. Although the phenomenon of the inducible splicing switch (or inducible exon) is well documented, the molecular underpinnings of these curious changes have remained mysterious. We describe methods to study how the glutamate NMDA receptor, which is a fundamental component of interneuronal signaling and plasticity, undergoes an inducible switch in its splicing pattern in primary neurons. This splicing switch promotes the skipping of an exon that encodes the CI cassette protein module, which is thought to communicate signals from the membrane to the cell nucleus during neuronal activity. We show that this induced splicing event is regulated in neurons by a three-part (UAGG-type) sequence code for exon silencing, and demonstrate a wider role for exon-skipping responsiveness in transcripts with known synaptic functions that also harbor a similar sequence code.
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影响因子：
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