Determinants that regulate splicing of SMN

描述(由申请人提供)： 脊髓性肌萎缩症(SMA)由端粒5q13缺失引起 SMN1基因的拷贝。着丝粒基因5q13 SMN2编码一个相同的 蛋白。虽然SMN2的损失并不会导致SMA的发展，但 SMN2的存在在体内以剂量依赖的方式作为疾病修饰物 没有SMN1。SMN1基因产生一个全长转录本，而 SMN2的主要产物是一个没有外显子7的外显子跳过的mRNA。 SMN1和SMN2之间的单个非多态核苷酸差异是 这是这种替代拼接的基础。通过比较衍生的杂交SMN基因 从有未受影响亲属的SMA患者中，我们证明了 这一核苷酸(SMN1或SMN2)决定了临床结果。因此， SMA的临床演变与SMN基因的剪接有关。SMN 蛋白质自结合，蛋白质从另一种 拼接的SMN2RNA，形成低聚物的能力降低，稳定性较差 与全长蛋白质相比。此外，来自SMA的SMN点突变体 患者表现出与疾病严重程度成正比的有缺陷的寡聚化。 这些研究确定了发育的遗传和生化基础。 SMA。 着丝粒SMN2拷贝基因的主要缺陷是产生 外显子7跳过SMN蛋白。因为这个外显子的“包含”已经显示出来了 可能是SMA发病机制中最关键的分子决定因素 这项提案的重点将放在控制外显子7剪接的决定因素上。 目标1和目标2的目标是确定管理SMN的顺式要素 外显子7 RNA加工，包括外显子剪接增强子、抑制子、侧翼 内含子元件和外显子7内的临界C/T转变(SMN1=C； SMN_2=T)。这将通过广泛的遗传分析和 并辅之以体外剪接分析。延伸基因工作 在前两个目标中开发的目标3将识别和描述TRANS 调节SMN外显子7前-mRNA正确处理的因素。会是 确定因子与外显子7的结合是否与 刺激外显子7的包涵体。这些研究可能对开发 促进SMN-2来源的mRNAs中包含外显子7的治疗。

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) results from the loss of both telomeric 5q13 copies of the SMN1 gene. The centromeric 5q13 SMN2 gene encodes an identical protein. While the loss of SMN2 does not lead to the development of SMA, the presence of SMN2 acts as a disease modifier in a dose-dependent manner in the absence of SMN1. The SMN1 gene produces a full-length transcript, while the primary product of SMN2 is an exon-skipped mRNA lacking exon 7. We reported that a single non-polymorphic nucleotide difference between SMN1 and SMN2 is the basis for this alternative splicing. By comparing hybrid SMN genes derived from SMA patients with unaffected relatives, we demonstrated that the origin of this nucleotide (SMN1 or SMN2) dictated clinical outcome. Therefore, the clinical evolution of SMA correlates with the splicing of SMN genes. SMN protein self-associates, and the protein translated from the alternative spliced SMN2 RNA, has a reduced ability to form oligomers and is less-stable compared to the full-length protein. Furthermore, SMN point mutants from SMA patients show defective oligomerization proportional to their disease severity. These studies have defined the genetic and biochemical basis for development of SMA. The primary defect of the centromeric SMN2 copy gene is the production of the exon 7 skipped SMN protein. Since the "inclusion" of this exon has been shown to be perhaps the most critical molecular determinant in SMA pathogenesis, the focus of this proposal will be on the determinants that govern exon 7 splicing. The goals of Aims 1 and 2 are to identify the cis-elements that regulate SMN exon 7 RNA-processing, including exonic splice enhancers, silencers, flanking intronic elements, and the critical C/T transition within exon 7 (SMN1=C; SMN2=T). This will be accomplished by extensive genetic analysis and complemented by in vitro analysis of splicing. Extending the genetic work developed in the first two aims, Aim 3 will identify and characterize the trans factors that mediate proper processing of SMN exon 7 pre-mRNA. It will be determined whether factor binding to exon 7 correlates with the ability to stimulate exon 7 inclusion. These studies may be useful for the development of therapeutics that promote the inclusion of exon 7 in SMN2-derived mRNAs.