Determinants that regulate splicing of SMN

描述（由申请人提供）： 脊髓性肌萎缩症 (SMA) 是由 5q13 端粒缺失引起的 SMN1 基因的拷贝。着丝粒 5q13 SMN2 基因编码相同的 蛋白质。虽然 SMN2 的缺失不会导致 SMA 的发展， SMN2 的存在以剂量依赖性方式作为疾病调节剂 SMN1 缺失。 SMN1 基因产生全长转录本，而 SMN2 的主要产物是缺乏外显子 7 的外显子跳跃 mRNA。我们报道 SMN1和SMN2之间的单个非多态性核苷酸差异是 这种选择性剪接的基础。通过比较衍生的杂交SMN基因 从 SMA 患者及其未受影响的亲属中，我们证明了 该核苷酸（SMN1 或 SMN2）决定临床结果。因此， SMA 的临床进化与 SMN 基因的剪接相关。 SMN 蛋白质自缔合，并且蛋白质从替代物翻译而来 剪接的 SMN2 RNA，形成寡聚体的能力降低且稳定性较差 与全长蛋白质相比。此外，来自 SMA 的 SMN 点突变体 患者表现出与其疾病严重程度成比例的缺陷寡聚化。 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 跳过了 SMN 蛋白。由于该外显子的“包含”已被证明 也许是 SMA 发病机制中最关键的分子决定因素 该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 2 的目标是确定调节 SMN 的顺式元件 外显子 7 RNA 加工，包括外显子剪接增强子、沉默子、侧翼 内含子元件，以及外显子 7 内的关键 C/T 转变（SMN1=C； SMN2=T）。这将通过广泛的遗传分析和 辅以剪接的体外分析。扩展遗传工作 在前两个目标中制定的目标 3 将识别并描述反式 介导 SMN 外显子 7 前 mRNA 正确加工的因素。它将是 确定与外显子 7 结合的因子是否与以下能力相关： 刺激外显子 7 包含。这些研究可能有助于开发 促进 SMN2 衍生 mRNA 中包含外显子 7 的疗法。

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.