hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing
hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing
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DOI：
10.1093/hmg/ddm276
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发表时间：
2007-12-15
影响因子：
3.5
通讯作者：
Manley, James L.
中科院分区：
文献类型：
--
作者：
Kashima, Tsuyoshi;Rao, Nishta;Manley, James L.
运动神经元存活基因1（SMN 1）的纯合缺失或突变导致脊髓性肌萎缩症。SMN 1已在人体中复制以产生SMN 2，后者产生低水平的功能性SMN蛋白。然而，大多数SMN 2转录物缺乏外显子7，导致蛋白质无功能。外显子7 5'端附近的单核苷酸差异在很大程度上解释了SMN 2外显子7跳跃，这种效应归因于依赖于SR蛋白剪接因子ASF/SF 2的外显子剪接增强子（ESE）的丢失或通过剪接阻遏物hnRNP A1的结合发挥功能的外显子剪接沉默子（ESS）元件的产生。我们早期的实验有利于后者的机制，在这里，我们提供了进一步的证据支持ESS模型。我们证明了hnRNP A1缺失对SMN 2外显子7剪接的显著影响是特异性的，因为hnRNP A1缺失对测试的其他低效剪接事件几乎没有影响，并且ASF/SF 2缺失不影响SMN 1/2剪接。通过两种不同的方法，我们发现hnRNPA 1与SMN 2外显子7之间有很强的特异性相互作用，而ASF/SF 2与其他SR蛋白与SMN 1和SMN 2外显子7的5'端之间只有很弱的等效相互作用。最后，我们描述了两个疾病相关的外显子跳跃突变，创建hnRNP A1结合位点，但剪接可以恢复只有适度或根本没有hnRNP A1耗尽。总之，我们的研究结果提供了强有力的支持的想法，SMN 2外显子7剪接是由hnRNPA 1依赖性ESS抑制，但也表明，创造这样的元素是上下文依赖性的。
Homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1) causes spinal muscular atrophy. SMN1 has been duplicated in humans to create SMN2, which produces a low level of functional SMN protein. However, most SMN2 transcripts lack exon 7, resulting in a non-functional protein. A single nucleotide difference near the 5' end of exon 7 largely accounts for SMN2 exon 7 skipping, an effect that has been attributed to loss of an exonic splicing enhancer (ESE) dependent on the SR protein splicing factor ASF/SF2 or to the creation of an exonic splicing silencer (ESS) element that functions by binding of the splicing repressor hnRNP A1. Our earlier experiments favored the latter mechanism and here we provide further evidence supporting the ESS model. We demonstrate that the striking effect of hnRNP A1 depletion on SMN2 exon 7 splicing is specific, as hnRNP A1 depletion has little or no effect on other inefficient splicing events tested, and ASF/SF2 depletion does not affect SMN1/2 splicing. By two different methods, we find a strong and specific interaction of hnRNPA1 with SMN2 exon 7 and only weak and equivalent interactions between ASF/SF2 and other SR proteins with the 5' ends of SMN1 and SMN2 exon 7. Finally, we describe two disease-related exon-skipping mutations that create hnRNP A1 binding sites, but show that splicing can be restored only modestly or not at all by hnRNP A1 depletion. Together our results provide strong support for the idea that SMN2 exon 7 splicing is repressed by an hnRNPA1-dependent ESS, but also indicate that creation of such elements is context-dependent.