Depolarization and CaM kinase IV modulate NMDA receptor splicing through two essential RNA elements.
Depolarization and CaM kinase IV modulate NMDA receptor splicing through two essential RNA elements.
复制标题
去极化和CAM激酶IV通过两个必需的RNA元素调节NMDA受体剪接。
DOI：
10.1371/journal.pbio.0050040
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发表时间：
2007-02
期刊：
影响因子：
9.8
通讯作者：
Black, Douglas L
中科院分区：
文献类型：
--
作者：
Lee, Ji-Ann;Xing, Yi;Nguyen, David;Xie, Jiuyong;Lee, Christopher J;Black, Douglas L
选择性剪接控制许多对神经元兴奋很重要的蛋白质的活性，但影响剪接亚型表达的信号转导途径尚不清楚。一种特别有趣的选择性剪接系统是 NMDA 受体 1 (NMDAR1 E21) 的外显子 21 (E21)，它控制 NMDA 受体向质膜的运输，并受到 Ca++/钙调蛋白依赖性蛋白激酶 (CaMK) IV 信号传导的抑制。在这里，我们表征了 NMDAR1 E21 的剪接。我们发现 E21 剪接受到神经元去极化的可逆抑制，并且我们在外显子内识别出两个 RNA 元件，它们共同发挥作用来介导诱导性抑制。这些外显子元件之一类似于内含子 CaMK IV 响应性 RNA 元件 (CaRRE)，最初在 BK 通道 STREX 外显子的 3' 剪接位点中发现，但之前在外显子内未观察到。另一个元素是新的 RNA 基序。将这两个基序（称为 CaRRE 1 型和 CaRRE 2 型）中的任何一个引入异源组成型外显子中，都可以对新外显子产生 CaMK IV 依赖性抑制。因此，任一外显子 CaRRE 都足以抑制 CaMK IV 诱导的抑制。单核苷酸扫描诱变定义了这两个 CaRRE 基序的共有序列。全基因组基序搜索和随后的 RT-PCR 验证确定了一组携带 CaRRE 共有序列的去极化调节替代外显子。许多这些外显子可能会改变神经元功能。因此，这两个RNA元件定义了一组共同调节的剪接事件，这些剪接事件响应神经元中的共同刺激来改变其活动。 NMDA 受体 1 外显子 21 的选择性剪接在神经元中以 CaMK IV 依赖性方式被去极化可逆地抑制。这表明剪接是通过动态活动输入进行微调的。多种机制直接改变神经元活动以响应外部刺激，从膜蛋白的短效修饰到基因表达的长效变化。基因表达中经常受到调节的步骤是前 mRNA 剪接反应，其中包含外显子（蛋白质编码序列）或剪接位点的位置会产生编码功能不同蛋白质的选择性剪接 mRNA 亚型。在这里，我们研究 NMDA 受体的剪接，它响应神经递质谷氨酸来改变神经元活动。我们发现，NMDA 受体亚基 NR1 mRNA 中重要外显子 (E21) 的剪接受到细胞去极化和细胞内信号分子 CaMK IV 激活的抑制。我们发现这种剪接抑制是由外显子本身内的两个调节序列介导的。一个序列与先前描述的调节元件相似，但尚不清楚该元件在外显子中发挥作用。另一个是新元素。这些元件作为简并序列家族的特征使得能够鉴定出一组对细胞去极化和 CamK IV 具有共同反应性的外显子。这些结果定义了在调节神经元活动时可能发生的一组新的基因表达变化。
Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity. Alternative splicing of NMDA receptor 1 exon 21 is reversibly repressed by depolarization in a CaMK IV-dependent manner in neurons. This suggests splicing is finely tuned by dynamic activity inputs. Multiple mechanisms direct changes in neuronal activity in response to external stimuli, ranging from short-acting modifications of membrane proteins to longer-acting changes in gene expression. A frequently regulated step in gene expression is the pre-mRNA splicing reaction in which the inclusion of exons (protein-coding sequences) or the position of splice sites produces alternatively spliced mRNA isoforms encoding functionally different proteins. Here, we study splicing of the NMDA receptor, which responds to the neurotransmitter glutamate to modify neuronal activity. We show that the splicing of an important exon (E21) in the NMDA receptor subunit NR1 mRNA is repressed by cell depolarization and activation of the intracellular signaling molecule, CaMK IV. We find that this splicing repression is mediated by two regulatory sequences within the exon itself. One sequence is similar to a previously described regulatory element that had not been known to function in an exon. The other is a new element. The characterization of these elements as a family of degenerate sequences allowed the identification of a group of exons sharing responsiveness to cell depolarization and CamK IV. These results define a new set of gene expression changes that may occur in modulating neuronal activity.