Singh Natalia N, Androphy Elliot J, Singh Ravindra N
Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA.
Biochem Biophys Res Commun. 2004 Mar 5;315(2):381-8. doi: 10.1016/j.bbrc.2004.01.067.
SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.
SMN1和SMN2代表人类运动神经元存活基因的两个几乎相同的拷贝。脊髓性肌萎缩症（SMA）最常见的病因是SMN1缺失，同时由于外显子7第6位（C6U）的抑制性突变导致外显子7被排除，使得SMN2无法发挥补偿作用。这一单外显子核苷酸如何调节外显子7的识别一直是人们主要关注的问题。基于评分矩阵和体外实验，与SF2/ASF相关的外显子剪接增强子（ESE）的缺失被认为是外显子7被排除的原因。然而，最近的一份报告支持与hnRNP A1相关的外显子剪接沉默子（ESS）的产生是外显子7被排除的决定性因素。在这里，我们表明C6U加强了一个抑制性环境，该环境覆盖的序列比hnRNP A1结合位点更大。在SMN1外显子7的第一位添加一个G残基也可以加强这种抑制性环境，尽管存在SF2/ASF结合位点，但仍会促进外显子7跳跃。通过体内筛选和一系列突变，我们证明外显子7 5'端延伸的抑制性环境的加强是通过重叠的序列基序实现的，这些基序共同协作调节外显子的使用。
