In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes
In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes
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DOI：
10.1261/rna.7580704
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发表时间：
2004-08-01
期刊：
影响因子：
4.5
通讯作者：
Singh, RN
中科院分区：
文献类型：
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作者：
Singh, NN;Androphy, EJ;Singh, RN
人类有两个几乎相同的运动神经元(SMN)存活基因拷贝，SMN1和SMN2。在脊髓性肌萎缩症(SMA)中，SMN1不能补偿由于外显子7的第6位抑制性突变(转录本中的C6U突变)而导致的SMN1的丢失。我们最近发现，C6U产生了一种延长的抑制上下文(外显子)，导致SMN1的外显子7跳过。以前的研究表明，SMN1和SMN2中的外显子7都需要与Tra2相关的外显子剪接增强子(Tra2-ESE)。在这里，我们描述了确定野生型核苷酸在整个外显子7中的位置特异性作用的活体选择方法。我们的结果证实了外显子7 3‘端附近存在排除的存在，并揭示了在外显子7的3’端附近存在额外的抑制区(3‘-簇)。我们还证明了外显子7最后一个位置的单核苷酸替换改善了5’剪接位点(Ss)，因此抑制元件的存在(外显子和3‘-簇)的存在和Tra2-ESE的缺失不再决定外显子7的使用。我们的结果表明，进化保守的弱5‘s可能是在不同的生理环境下调节外显子7剪接的一种机制。这是第一个应用功能选择方法分析整个外显子的报告。这种方法在确定剪接位点的相对强度以及识别新的外显子顺式元件方面具有无与伦比的优势。
Humans have two near identical copies of the survival of motor neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to an inhibitory mutation at position 6 (C6U mutation in transcript) of exon 7. We have recently shown that C6U creates an extended inhibitory context (Exinct) that causes skipping of exon 7 in SMN2. Previous studies have shown that an exonic splicing enhancer associated with Tra2 (Tra2-ESE) is required for exon 7 inclusion in both SMN1 and SMN2. Here we describe the method of in vivo selection that determined the position-specific role of wild-type nucleotides within the entire exon 7. Our results confirmed the existence of Exinct and revealed the presence of an additional inhibitory tract (3'-Cluster) near the 3'-end of exon 7. We also demonstrate that a single nucleotide substitution at the last position of exon 7 improves the 5' splice site (ss) such that the presence of inhibitory elements (Exinct as well as the 3'-Cluster) and the absence of Tra2-ESE no longer determined exon 7 usage. Our results suggest that the evolutionary conserved weak 5' ss may serve as a mechanism to regulate exon 7 splicing under different physiological contexts. This is the first report in which a functional selection method has been applied to analyze the entire exon. This method offers unparallel advantage for determining the relative strength of splice sites, as well as for identifying the novel exonic cis-elements.