NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizure.
NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizure.
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DOI：
10.7554/elife.00178
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发表时间：
2013-01-22
期刊：
影响因子：
7.7
通讯作者：
Darnell RB
中科院分区：
文献类型：
--
作者：
Eom T;Zhang C;Wang H;Lay K;Fak J;Noebels JL;Darnell RB
神经元RNA结合蛋白NOVA调节剪接，穿梭于细胞质，并与树突中的靶转录物共定位，表明剪接和局部翻译之间的联系。在这里，我们鉴定了超过200个转录物，这些转录物显示出大量的NOVA依赖性变化，但令人惊讶的是，HITS-CLIP显示NOVA在内含子而不是3′ UTR中结合这些RNA。这使我们发现了这些内含子内的秘密外显子的NOVA调节剪接。这些外显子触发了无义介导的衰变（NMD），因为NOVA对RNA水平的影响需要UPF 1和蛋白质合成。它们的调节是动态的和生理相关的。NMD外显子受癫痫发作的调节，癫痫发作也诱导了Nova亚细胞定位的变化，并介导了突触蛋白（包括与家族性癫痫有关的蛋白）的巨大变化。此外，Nova单倍不足小鼠具有自发性癫痫。这些数据揭示了一种隐藏的动态RNA调节手段，将电活性与剪接和蛋白质输出联系起来，并介导神经元中的稳态兴奋/抑制平衡。DOI：http://dx.doi.org/10.7554/eLife.00178.001在基因中的DNA被转录成信使RNA后，称为内含子的mRNA部分被去除，而mRNA的其余部分，即外显子，被拼接在一起。在真核细胞内，一种称为选择性剪接的过程允许单个基因编码多种蛋白质变体，确保某些外显子包含在最终修饰的mRNA中，而其他外显子被排除在外。然后，这种修饰的mRNA被翻译成蛋白质。真核细胞还含有与RNA结合以调节可变剪接的蛋白质。这些RNA结合蛋白通常存在于细胞的细胞质和细胞核中，它们参与剪接可能与细胞中的其他过程有关，如mRNA定位和翻译。在过去的二十年里，人们也越来越清楚地发现，某些类型的RNA结合蛋白，包括NOVA蛋白，只在神经元中发现，这些蛋白质已被最好地表征为选择性剪接调节剂。最近的研究还表明，它们在调节神经元活动和发育中也具有重要作用，并且它们在神经元细胞核和细胞质中的作用可能是协调的。现在，Eom等人使用称为HITS-CLIP的高通量测序和交联方法的预测能力来表明NOVA蛋白可以通过调节细胞核中的选择性剪接过程来间接调节细胞质mRNA水平，以在小鼠大脑中产生“隐藏”外显子。mRNA中这些外显子的存在导致细胞质中提前终止密码子的产生。这些密码子触发了一个被称为无义介导的衰变过程，该过程涉及识别含有无义突变的mRNA转录本，然后降解它们。这些隐藏的外显子在缺失NOVA蛋白的小鼠中观察到，在那里它们以异常高的水平表达;在正常小鼠中，这些外显子以前从未见过，因此它们被称为“隐藏的”。Eom等人还表明，这些隐性外显子通过诱导小鼠癫痫发作而具有生理相关性。癫痫发作后，他们发现NOVA蛋白上调和下调不同隐蔽外显子的水平，导致这些mRNA编码的蛋白质水平发生变化，包括抑制进一步癫痫发作的蛋白质。总的来说，这些结果表明，通过控制神经元中各种蛋白质的产生，这些以前未知的神秘外显子在大脑的工作中发挥着重要作用。DOI：http://dx.doi.org/10.7554/eLife.00178.002网站
The neuronal RNA binding protein NOVA regulates splicing, shuttles to the cytoplasm, and co-localizes with target transcripts in dendrites, suggesting links between splicing and local translation. Here we identified >200 transcripts showing NOVA-dependent changes in abundance, but, surprisingly, HITS-CLIP revealed NOVA binds these RNAs in introns rather than 3′ UTRs. This led us to discover NOVA-regulated splicing of cryptic exons within these introns. These exons triggered nonsense mediated decay (NMD), as UPF1 and protein synthesis were required for NOVA's effect on RNA levels. Their regulation was dynamic and physiologically relevant. The NMD exons were regulated by seizures, which also induced changes in Nova subcellular localization and mediated large changes in synaptic proteins, including proteins implicated in familial epilepsy. Moreover, Nova haploinsufficient mice had spontaneous epilepsy. The data reveal a hidden means of dynamic RNA regulation linking electrical activity to splicing and protein output, and of mediating homeostatic excitation/inhibition balance in neurons. DOI: http://dx.doi.org/10.7554/eLife.00178.001 After the DNA in a gene has been transcribed into messenger RNA, portions of the mRNA called introns are removed, and the remaining stretches of mRNA, which are known as exons, are spliced together. Within eukaryotic cells, a process known as alternative splicing allows a single gene to encode for multiple protein variants by ensuring that some exons are included in the final, modified mRNA, while other exons are excluded. This modified mRNA is then translated into proteins. Eukaryotic cells also contain proteins that bind to RNA to regulate alternative splicing. These RNA-binding proteins are often found in both the cytoplasm and nucleus of cells, and their involvement in splicing may be linked to other processes in the cell such as mRNA localization and translation. It has also become clear over the past two decades that certain types of RNA-binding proteins, including NOVA proteins, are only found in neurons, and that these proteins have been best characterized as alternative splicing regulators. Recent work has also suggested that they also have important roles in regulating neuronal activity and development, and that their actions in neuronal nuclei and cytoplasm might be coordinated. Now Eom et al. use the predictive power of a high throughput sequencing and crosslinking method termed HITS-CLIP to show that NOVA proteins can indirectly regulate cytoplasmic mRNA levels by regulating the process of alternative splicing in the nucleus to produce ‘cryptic’ exons in the brains of mice. The presence of these exons in the mRNA leads to the production of premature termination codons in the cytoplasm. These codons trigger a process called nonsense-mediated decay that involves identifying mRNA transcripts that contain nonsense mutations, and then degrading them. These cryptic exons were seen in mice missing the NOVA proteins, where they are expressed in abnormally high levels; in normal mice, these exons have not been seen before, hence they were termed ‘cryptic’. Eom et al. also show that these cryptic exons are physiologically relevant by inducing epileptic seizures in mice. Following the seizures, they find that the NOVA proteins up-regulate and down-regulate the levels of different cryptic exons, leading to changes in the levels of the proteins encoded by these mRNAs, including proteins that inhibit further seizures. Overall the results indicate that, by controlling the production of various proteins in neurons, these previously unknown cryptic exons have important roles in the workings of the brain. DOI: http://dx.doi.org/10.7554/eLife.00178.002