Determinants that regulate splicing of SMN

描述（由申请人提供）： 脊髓性肌萎缩症（SMA）是由于端粒5q13和5q14的缺失引起的。 SMN1基因的复制。着丝粒5q13 SMN2基因编码一个相同的 蛋白虽然SMN2的缺失不会导致SMA的发展，但 SMN2的存在以剂量依赖性方式作为疾病修饰因子， 没有SMN1。SMN1基因产生全长转录本，而 SMN2的主要产物是缺少外显子7的外显子跳过的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。通过比较杂交SMN基因， 从SMA患者与未受影响的亲属，我们证明， 该核苷酸（SMN1或SMN2）决定了临床结果。因此 SMA的临床演变与SMN基因的剪接相关。SMN 蛋白质自缔合，并且从替代物翻译的蛋白质 剪接的SMN2 RNA，形成寡聚体的能力降低，稳定性降低 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寡聚化缺陷。 这些研究已经确定了发展的遗传和生物化学基础， SMA。 着丝粒SMN2拷贝基因的主要缺陷是产生 外显子7跳读SMN蛋白。由于该外显子的"包含"已被证明 可能是SMA发病机制中最关键的分子决定因素， 该建议的重点将是控制外显子7剪接的决定因素。 目的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 内含子元件和外显子7内的关键C/T转换（SMN1 = C; SMN2 = T）。这将通过广泛的遗传分析来完成， 并辅以体外剪接分析。扩展遗传工作 在前两个目标中，目标3将确定和描述跨 介导SMN外显子7前体mRNA正确加工的因素。将 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的治疗剂。

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.